Peptides promoting lipid efflux via abca1 and activating a lipoprotein lipase

ABSTRACT

Disclosed herein are peptides and peptide analogs with multiple amphipathic α-helical domains that promote lipid efflux from cells via an ABCA1-dependent pathway, as well as peptides that activate lipoprotein lipase, and compositions comprising such peptides or combinations thereof. Also provided herein are methods of using multi-domain amphipathic α-helical peptides or peptide analogs to treat or inhibit dyslipidemic disorders. Methods for identifying non-cytotoxic peptides that promote ABCA1-dependent lipid efflux from cells and activate lipoprotein lipase within cells are also disclosed herein.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/045,213, filed Apr. 15, 2008, which is incorporated herein byreference in its entirety.

FIELD

This disclosure relates to peptides or peptide analogs with multipleamphipathic α-helical domains that promote lipid efflux from cells viaan ABCA1-dependent pathway and activate lipoprotein lipase. Thedisclosure further relates to methods for characterizing multi-domainamphipathic α-helical peptides that promote lipid efflux from cells andactivate lipoprotein lipase in cells. Multi-domain amphipathic α-helicalpeptides that promote lipid efflux from cells via an ABCA1-dependentpathway in a substantially non-cytotoxic manner (e.g., without causingsignificant hypertriglyceridemia) are useful in the treatment andprevention of dyslipidemic and vascular disorders.

BACKGROUND

Clearance of excess cholesterol from cells by high density lipoproteins(HDL) is facilitated by the interaction of HDL apolipoprotein withcell-surface binding sites or receptors (Mendez et al., J. Clin. Invest.94:1698-1705, 1994), such as ABCA1 (Oram and Yokoyama, J. Lipid Res.37:2473-2491, 1996). ABCA1 is a member of the ATP binding cassettetransporter family (Dean and Chimini., J. Lipid Res. 42:1007-1017, 2001)and is expressed by many cell types (Langmann et al., Biochem. Biophys.Res. Commun. 257:29-33, 1999). Mutations in the ABCA1 transporter leadto Tangier disease, which is characterized by the accumulation of excesscellular cholesterol, low levels of HDL and an increased risk forcardiovascular disease (Rust et al., Nat. Genet. 22:352-355, 1999;Bodzioch et al., Nat. Genet. 22:347-351, 1999; Brooks-Wilson et al.,Nat. Genet. 22:336-345, 1999; Remaley et al., Proc. Natl. Acad. Sci. USA96:12685-12690, 1999; and Lawn et al., J. Clin. Invest. 104:R25—R31,1999). Fibroblasts from Tangier disease patients are defective in theinitial step of cholesterol and phospholipid efflux to extracellularapolipoproteins (Francis et al., J. Clin. Invest. 96:78-87, 1995 andRemaley et al., Arterioscler. Thromb. Vasc. Biol. 17:1813-1821, 1997).

Research has demonstrated an inverse correlation between the occurrenceof atherosclerosis events and levels of HDL and its most abundantprotein constituent, apolipoprotein A-I (apoA-I) (Panagotopulos et al.,J. Biol. Chem. 277:39477-39484, 2002). ApoA-I has been shown to promotelipid efflux from ABCA1-transfected cells (Wang et al., J. Biol. Chem.275:33053-33058, 2000; Hamon et al., Nat. Cell Biol. 2:399-406, 2000;and Remaley et al., Biochem. Biophys. Res. Commun. 280:818-823, 2001).However, the nature of the interaction between apoA-I and ABCA1 is notfully understood. Several other exchangeable-type apolipoproteins havealso been shown to efflux lipid from ABCA1-transfected cells (Remaley etal., Biochem. Biophys. Res. Commun. 280:818-823, 2001). Although theexchangeable-type apolipoproteins do not share a similar primary aminoacid sequence, they all contain amphipathic helices, a structural motifknown to facilitate the interaction of proteins with lipids (Segrest etal., J. Lipid Res. 33:141-166, 1992 and Anantharamaiah et al., J. Biol.Chem. 260:10248-10255, 1985). Animal experiments have shown thatintravenous injections of apoA-I or its variant, apoA-I Milano (whichhas a cysteine substitution at position 173 for arginine), producedsignificant regression of atherosclerosis (Rubin et al., Nature353:265-267, 1991 and Nissen et al., JAMA 290:2292-2300, 2003). Theseresults make apoA-1, or derivatives thereof, attractive as potentialtherapeutic compounds in the treatment and prevention ofatherosclerosis.

Short synthetic peptide mimics of apolipoproteins have been used as amodel for studying physical and biological properties of apolipoproteins(see, e.g., Fukushima et al., J. Am. Chem. Soc. 101:3703-3704, 1980;Kanellis et al., J. Biol. Chem. 255:11464-11472, 1980; and U.S. Pat.Nos. 4,643,988, and 6,376,464). These include, for instance, singlehelices taken from native apolipoproteins, synthetic amphipathic alphahelices (Kanellis et al., J. Biol. Chem. 255:11464-11472, 1980), andderivatives thereof. Examples of short synthetic amphipathic helicalpeptides have been shown to promote lipid efflux and inhibitatherosclerosis (Garber et al, J. Lipid Res. 42:545-552, 2001; Navab etal., Circulation 105:290-292, 2002; and U.S. Pat. No. 6,156,727).However, while some of these peptides exhibit beneficial effects inpreventing atherosclerosis, they are also potentially cytotoxic (Remaleyet al., J. Lipid Res. 44:828-836, 2003). It is believed that thecytotoxicity is caused by non-specific, ABCA1-independent lipid effluxfrom cells (Remaley et al., J. Lipid Res. 44:828-836, 2003). Therefore,there exists a need for non-cytotoxic synthetic peptide mimics ofapolipoproteins that promote specific lipid efflux from cells by anABCA1-dependent pathway for use in the treatment and prevention ofcardiovascular diseases, such as atherosclerosis.

SUMMARY OF THE DISCLOSURE

Isolated peptides and peptide analogs including peptides with multipleamphipathic α-helical domains that promote lipid efflux from cells viaan ABCA1-dependent pathway have been identified and are describedherein. In various embodiments, a first amphipathic α-helical domainexhibits higher lipid affinity relative to a second amphipathicα-helical domain in the same peptide. In one example, the multi-domainpeptide includes two amphipathic α-helical domains and the peptidecomprises an amino acid sequence as set forth in any one of SEQ ID NOs:3-45.

Also disclosed herein are isolated peptides and peptide analogs capableof promoting lipid efflux from cells by an ABCA1-dependent pathway whichinclude a domain capable of activating lipoprotein lipase. In oneembodiment, a peptide comprises a first domain that is an amphipathicα-helical domain that promotes lipid efflux from cells by anABCA1-dependent pathway, and a second domain including an apoC-IIprotein, or a variant or fragment thereof that retains a lipoproteinlipase-activating property. For example, in some instances the firstdomain is an amphipathic α-helical domain with a hydrophobic momentscore (Eisenberg scale; 100 degree-alpha helix) per residue of about0.34 to about 0.60 and the second domain comprises an ApoC-II proteinwith an amino acid sequence set forth in any one of SEQ ID NOs: 63-69.In certain embodiments, the peptide or peptide analog includes an aminoacid sequence as set forth in any one of SEQ ID NOs: 70-75. In otherembodiments, these domains are provided in a single preparation (ormixture or combination of preparations), but the domains are not allcovalently linked to each other; e.g., the domain capable of activatinglipoprotein lipase is on a different peptide or protein than thedomain(s) capable of promoting lipid efflux from cells by anABAC1-dependent pathway.

Also described herein is a method of treating dyslipidemic and vasculardisorders in a subject, including administering to the subject atherapeutically effective amount of the isolated multi-domain peptidesor peptide analogs described herein. Dyslipidemic and vascular disordersamenable to treatment with the isolated multi-domain peptides disclosedherein include, but are not limited to, hyperlipidemia,hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDLdeficiency, apoA-I deficiency, coronary artery disease, atherosclerosis,thrombotic stroke, peripheral vascular disease, restenosis, acutecoronary syndrome, reperfusion myocardial injury, vasculitis,inflammation, or combinations of two or more thereof. In variousembodiments, a peptide (or mixture of peptides) including a first domainwith ABCA-1 dependent lipid efflux activity and a second domain withlipoprotein lipase activity is administered at a therapeuticallyeffective concentration to prevent, inhibit or reduce a dyslipidemic orvascular disorder. In some embodiments, one of the disclosed peptidescomprising an amino acid sequence set forth in any one of SEQ ID NOs:70-75 is administered at a therapeutically effective concentration toprevent, inhibit or reduce a dylipidemic or vascular disorder. In otherembodiments, one of the disclosed peptides including ABCA-1 dependentlipid efflux activity, such as a peptide comprising an amino acidsequence set forth in any one of SEQ ID NOs: 3-45, is administered at atherapeutically effective concentration in combination with atherapeutically effective concentration of a lipoprotein lipaseactivator, such as a peptide comprising an amino acid sequence set forthin any one of SEQ ID NOs:63-69, to prevent, inhibit or reduce adylipidemic or vascular disorder. Optionally, such peptides areadministered in combination with a peptide (or analog) that activateslipoprotein lipase. Representative lipoprotein lipase activatingpeptides are described.

A method for identifying substantially non-cytotoxic peptides thatpromotes ABCA1-dependent lipid efflux from cells and/or are capable ofactivating lipoprotein lipase is also described. In various embodiments,the method includes performing one of more cytotoxicity tests, one ormore lipid efflux tests, and one or more lipoprotein lipase activitytests on ABCA1/liproprotein lipase-expressing and non-ABCA1/lipoproteinlipase-expressing cells, thereby identifying one or more substantiallynon-cytotoxic peptides that promote ABCA1-dependent lipid efflux fromcells and are capable of activating lipoprotein lipase in such cells.Example peptides for use in methods for identifying substantiallynon-cytotoxic peptides that promote ABCA1-dependent efflux from cellsinclude peptides that contain two or more domains, such as anamphipathic α-helical domain that promotes lipid efflux from cells by anABCA1-dependent pathway, and a ApoC-II protein domain, or a variant orfragment thereof that retains a lipoprotein lipase-activating property.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F are a set of graphs illustrating lipid efflux by ABCA1transfected cells and control cells treated with various peptides. ABCA1transfected cells (closed circle) and control cells (open circle) weregrown in alpha-MEM media with 10% FCS and were examined for theirability to efflux cholesterol (FIGS. 1A, 1C and 1E) and phospholipid(FIGS. 1B, 1D and 1F) over 18 hours to apoA-I (FIGS. 1A and 1B), L-37pA(FIGS. 1C and 1D), and D-37pA (FIGS. 1E and 1F). Results are expressedas the mean of triplicates ±1 SD.

FIGS. 2A-2B are a pair of graphs illustrating the time course for lipidefflux by ABCA1 transfected cells and control cells treated with apoA-Iand L-37pA. Cholesterol efflux from either ABCA1 transfected cells (FIG.2A) or control cells (FIG. 2B) treated with 10 μg/ml apoA-I (square), 10μg/ml L-37pA peptide (triangle), and blank media (circle) (α-MEM plus 1mg/ml BSA) was determined at the time points indicated on the x axis.Results are expressed as the mean of triplicates ±1 SD.

FIG. 3 is a graph illustrating solubilization of DMPC vesicles bysynthetic peptides. The indicated peptides (L-37pA (L), D-37pA (D),L2D-37pA (L2D), L3D-37pA (L3D), and apoA-I (A)) at a final concentrationof 0.4 mg/ml were incubated with DMPC vesicles (2 mg/ml) for 2 hours andthe decrease in turbidity (indicative of vesicle lysis) was monitored atan absorbance of 350 nm. Results are expressed as the mean oftriplicates ±1 SD.

FIGS. 4A-4B are a pair of graphs illustrating lipid efflux by ABCA1transfected cells and control cells treated with mixed L- and D-aminoacid 37pA peptides. ABCA1 transfected cells (closed symbols) and controlcells (open symbols) were examined for their ability to effluxcholesterol (FIG. 4A) and phospholipid (FIG. 4B) over an 18 hour periodwhen treated with 10 ng/ml L2D-37pA (closed circle, open circle) and 10μg/ml L3D-37pA (closed square, open square). Results are expressed asthe mean of triplicates ±1 SD.

FIG. 5 is a graph illustrating ABCA1-independent efflux of cholesterolfrom Tangier disease fibroblasts. Normal skin fibroblasts (open bars)and Tangier disease skin fibroblasts (solid bars) were examined fortheir ability to efflux cholesterol over an 18 hour period when treatedwith 10 μg/ml apoA-I (A), 10 μg/ml L-37pA (L), and 10 μg/ml D-37pA (D).Results are expressed as the mean of triplicates ±1 SD.

FIGS. 6A-6B are a pair of graphs illustrating the effect of cellfixation on cholesterol efflux from ABCA1 transfected cells and controlcells. ABCA1 transfected cells (FIG. 6A) and control cells (FIG. 6B)were examined for their ability to efflux cholesterol when treated withapoA-I (A), L-37pA (L), D-37pA (D), and (0.02%) taurodeoxycholate (T)before (open bars) and after (solid bars) fixation with 3%paraformaldehyde. Synthetic peptides and apoA-I were used at aconcentration of 10 μg/ml, and cholesterol efflux was measured after 18hours. Efflux due to taurodeoxycholate treatment was measured after 1hour. Results are expressed as the mean of triplicates ±1 SD.

FIGS. 7A-7B are a pair of graphs illustrating the competitive binding ofL-37pA peptide to ABCA1 transfected cells and control cells. ABCA1 cells(FIG. 7A) and control cells (FIG. 7B) were incubated for 3 hours at 4°C. with the indicated concentration of the competitor proteins [L-37pA(triangle), D-37pA (open square), apoA-I (closed circle), L2D-37pA(star), and L3D-37pA (open circle)] and were then washed and incubatedfor 1 hour at 4° C. with 1 μg/ml of radiolabeled L-37pA peptide. Resultsare expressed as the mean of triplicates ±1 SD.

FIG. 8 is a graph plotting the calculated hydrophobic moment of the 37pApeptide and derivative peptides (1A, 2A, 3A, 4A, 5A, and 10A) with theirretention time on a reverse phase HPLC. Approximately 1 mg of each ofthe peptides was injected on a C-18 reverse phase HPLC column and elutedwith 25-85% gradient of acetonitrile containing 0.1% TFA.

FIG. 9 is a graph illustrating red blood cell lysis by the 37pA peptideand derivative peptides (1A, 2A, 3A, 4A, 5A, and 10A). Red blood cellswere incubated with the indicated concentration of the peptides for 1hour at 37° C. Results are expressed as the mean of triplicates ±1 SD.

FIGS. 10A-10F are a set of graphs illustrating cholesterol efflux byABCA1 transfected cells and control cells when treated with the 37pApeptide and derivative peptides (37pA, FIG. 10A; 1A, FIG. 10B; 2A, FIG.10C; 3A, FIG. 10D; 4A, FIG. 10E; and 5A, FIG. 10F). ABCA1 transfectedcells (grey squares) and control cells (solid triangles) were examinedfor their ability to efflux cholesterol over an 18 hour period whentreated with the indicated concentration of peptide. ABCA1-specificefflux was calculated by subtracting the cholesterol efflux results fromthe ABCA1 transfected cells from the control cells (open diamonds).Results are expressed as the mean of triplicates ±1 SD.

FIG. 11 is a graph illustrating 5A-POPC (1-palmitoyl-2-oleoylphosphatidylcholine peptide-mediated alterations in plasma cholesterolafter IV infusion of increasing doses of 5A peptide in wild-type miceover 24 hours. Increasing doses of the peptide resulted in increasedplasma HDL as result of ABCA-1 mediated efflux.

FIG. 12 is a graph illustrating atherosclerosis measurement in ApoE KOmice treated for 12 weeks with 5A peptide versus POPC control. Asignificant difference of 29% was observed in female mice that received30 mg/kg of peptide. This dose showed only modest changes in cholesterolefflux to plasma (see FIG. 11), suggesting that higher doses of thepeptide could lead to further reduction and possibly regression ofplaques.

FIG. 13 is a series of graphs illustrating changes in liver enzymes,hemolytic parameter, kidney function and glucose levels in wild-typemice receiving increasing doses of the 5A peptide. No significantchanges were observed in the measured parameters, including weightduring the study. Some of the parameters did show improvement comparedto control and baseline.

FIG. 14 is a graph illustrating the change in plasma triglycerides afterintravenous (IV) infusion of increasing doses of 5A-POPC peptide over 24hours, after over night fasting in wild-type mice. The increasing dosesof the 5A-POPC peptide led to a proportional increase in triglyceridelevels, up to 3 fold the baseline value. This effect has also beenobserved after the injection of the native ApoA-I lipoprotein.

FIG. 15 is a graph illustrating the change in plasma triglycerides afterIV infusion of increasing doses of 5A-POPC peptide over 24 hours, innon-fasting mice. Unlike a maximal 3 fold increase in plasmatriglycerides, when mice were not fasting an increase of up to 20 timeswas observed. No signs of toxicity were observed in mice receiving up to240 mg/kg of peptide during the study.

FIG. 16 is a graph illustrating triglyceride synthesis in mice afterlipase inhibition by Triton-WR 1339. Increasing doses of the 5A peptideled to no significant changes in triglycerides synthesis suggesting thatthe hypertriglyceridemia observed after 5A injection resulted fromdecreased catabolism of ApoB-containing lipoproteins.

FIG. 17 is a graph illustrating in vitro activity of lipoprotein lipaseat increasing doses of 5A peptide. Compared to baseline, the activity ofLPL decreased dramatically as the concentration of 5A-POPC peptideincreased.

FIG. 18 is a graph illustrating the ability of 18A-C-II:POPC peptide(peptide with SEQ ID NO: 70) to prevent hypertriglyceridemia. The18A-C-II:POPC peptide was lipidated with POPC at the ratio of 1:7.Hypertriglyceridemia was induced in C57B1/6 mice by a 2 mg (60 mg/kg)bolus intravenous (IV) injection of free 5A peptide (A). When 1 mg (30mg/kg) of the 18A-C-II peptide was co-injected with free 5A peptide (X),the increase in plasma triglycerides was significantly halted. Inaddition, a single IV dose of 1 mg of the 18A-C-II:POPC peptide loweredthe plasma triglyceride levels in normal C57B1 mice (□) compared tosaline (⋄).

FIG. 19 is a graph illustrating the effect of 18A-C-II:POPC (1:7)peptide on triglycerides of ApoE knockout mice. A single dose of 30mg/kg (1 mg) of 18A-C-II:POPC peptide significantly reduced plasmatriglyceride levels in apoE KO mice (76%) when compared to baselinevalues.

FIG. 20 is a graph illustrating the effect of 18A-C-II peptide on plasmacholesterol in ApoE KO mice. In order to investigate the effects of the18A-C-II peptide in plasma cholesterol of C57B1/6 mice, mice wereinjected with an IV bolus of 1 mg (30 mg/kg) of 18A-C-II:POPC and plasmacholesterol levels were monitored for 4 hours. A significant reductionof 52% and 80% was observed after 1 hour and 4 hours.

FIG. 21 is a graph illustrating the effect of 18A-C-II peptide on plasmacholesterol. ApoE knockout and LDLR knockout mice are characterized byhigh plasma levels of VLDL/IDL and LDL respectively. To evaluate thepotential to reduce the plasma cholesterol levels, mice were injected IVin bolus with a dose of 1 mg (30 mg/kg) of the lipidated 18A-C-II:POPCin ApoE KO (⋄) and 0.5 mg (15 mg/kg) in LDLR KO mice (□). A significantreduction (36%) in serum cholesterol in ApoE KO mice was observed, whilethe low dose of the peptide in LDLR KO mice showed a reduction of 22% inplasma cholesterol after 2 hours of the treatment.

FIGS. 22A-22B are graphs illustrating the effect of 18A-C-II peptide onplasma lipoproteins. The changes observed on plasma cholesterol andtriglycerides were further analyzed by fast protein liquidchromatography (FPLC). A dose of 30 mg/kg of 18A-C-II:POPC (1:7) peptidelead to reduction of LDL-c, HDL-c (left panel) as well as VLDL-tg,LDL-tg and HDL-tg (right panel) at one and four hours after treatment.(LDL-c=LDL cholesterol; tg=triglycerides). Each sample represents a poolof 3 mice.

FIG. 23 is a graph illustrating the effect of 18A-C-II peptide oncholesterol efflux. BHK and BHK-ABCA1 cells were loaded with³H-cholesterol for 24 hours at 37° C. and then 20 ug/ml of the free18A-C-II peptide was added as cholesterol acceptor to efflux for 18hours. Percentage efflux was calculated by subtracting the radioactivecounts in the blank from the radioactive counts in the presence of anacceptor and then dividing the result by the sum of the radioactivecounts in the medium plus the cell fraction. A significant differencewas observed on cells that express the ABCA1 transporter indicating thatABCA1 is, at least, one mechanism mediating the cholesterol efflux tothe 18A-C-II peptide.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. §1.822. In the accompanying sequence listing:

SEQ ID NO: 1 shows the amino acid sequence of the 37pA peptide.

SEQ ID NO: 2 shows the amino acid sequence of the gamma crystallinepeptide.

SEQ ID NOs: 3-45 show the amino acid sequences of a series of peptideswith apoA-1-like activity; these are also discussed in Table 1.

SEQ ID NOs: 46-49 show the amino acid sequences of several cellrecognition sequences.

SEQ ID NOs: 50-53 show the amino acid sequences of several cellinternalization sequences.

SEQ ID NO: 54 shows the amino acid sequence of a neutral cholesterolesterase activation sequence.

SEQ ID NO: 55 shows the amino acid sequence of an ACAT inhibitionsequence.

SEQ ID NOs: 56 and 57 show the amino acid sequences of a pair of LDLreceptor sequences.

SEQ ID NOs: 58-60 show the amino acid sequences of several anti-oxidantsequences.

SEQ ID NOs: 61 and 62 show the amino acid sequences of a pair of metalchelation sequences.

SEQ ID NOs: 63-69 show the amino acid sequences of a series of apoC-IIdomains with apoC-II-like activity (e.g., lipoprotein lipase activatingactivity); these are also discussed in Tables 3 and 5.

SEQ ID NOs: 70-75 show the amino acid sequences of a series of peptideswith apoA-1-like activity (e.g., promoting lipid efflux) and apoC-IIlike activity (e.g., lipoprotein lipase activating activity); these arealso discussed in Table 4.

DETAILED DESCRIPTION I. Abbreviations

ABCA1: ATP-binding cassette transporter A1

apoA-I: apolipoprotein A-I

apoC-II apolipoprotein C-II

DMPC: dimyristoyl phosphatidyl choline

FPLC: fast protein liquid chromatography

HDL: high-density lipoprotein

HPLC: high-pressure liquid chromatography

IV: intravenous

LDL: low-density lipoprotein

LPL: lipoprotein lipase

RBC: red blood cell

VLDL: very low density lipoprotein

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control.The materials, methods and examples are illustrative only and notintended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Alkane: A type of hydrocarbon, in which the molecule has the maximumpossible number of hydrogen atoms, and therefore has no double bonds(i.e., they are saturated). The generic formula for acyclic alkanes,also known as aliphatic hydrocarbons is C_(n)H_(2n+2); the simplestpossible alkane is methane (CH₄).

Alkyl group: refers to a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A “loweralkyl” group is a saturated branched or unbranched hydrocarbon havingfrom 1 to 10 carbon atoms.

Amphipathic: An amphipathic molecule contains both hydrophobic(non-polar) and hydrophilic (polar) groups. The hydrophobic group can bean alkyl group, such as a long carbon chain, for example, with theformula: CH₃(CH₂)_(n), (where n is generally greater than or equal toabout 4 to about 16). Such carbon chains also optionally comprise one ormore branches, wherein a hydrogen is replaced with an aliphatic moiety,such as an alkyl group. A hydrophobic group also can comprise an arylgroup. The hydrophilic group can be one or more of the following: anionic molecule, such as an anionic molecule (e.g., a fatty acid, asulfate or a sulfonate) or a cationic molecule, an amphoteric molecule(e.g., a phospholipid), or a non-ionic molecule (e.g., a small polymer).

One example of an amphipathic molecule is an amphipathic peptide. Anamphipathic peptide can also be described as a helical peptide that hashydrophilic amino acid residues on one face of the helix and hydrophobicamino acid residues on the opposite face. Optionally, peptides describedherein will form amphipathic helices in a physiological environment,such as for instance in the presence of lipid or a lipid interface.

Analog, derivative or mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

As used herein, the term “antibody” includes intact immunoglobulins aswell as a number of well-characterized fragments. For instance, Fabs,Fvs, and single-chain Fvs (SCFvs) that bind to target protein (orepitope within a protein or fusion protein) would also be specificbinding agents for that protein (or epitope). These antibody fragmentsare as follows: (1) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule produced by digestionof whole antibody with the enzyme papain to yield an intact light chainand a portion of one heavy chain; (2) Fab′, the fragment of an antibodymolecule obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody, a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and compositions of this disclosurecan be monoclonal or polyclonal. Merely by way of example, monoclonalantibodies can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-97, 1975) orderivative methods thereof. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Apolipoprotein C-II (apoC-II): A 79 amino acid protein, which plays arole in plasma lipid metabolism as an activator of lipoprotein lipase(LPL). This protein includes three amphipathic helices: helix 1,residues 16-38; helix 2, residues 45-58; and helix 3, residues 64-74.The lipase-activating region of apoC-II has previously been localized tothe C-terminal domain of the sequence, from about residue 56, whereasthe N-terminal domain (residues 1-50) of the sequence is involved inlipid binding.

In particular examples, apoC-II protein, fragment or variant thereof iscapable of activating lipoprotein lipase. For example, an apoC-IIprotein, fragment or variant thereof is administered with apoA-1 orpeptide mimics thereof to prevent, reduce or inhibithypertriglyceridemia. Unless the context clearly indicates otherwise,the term apoC-II includes any apoC-II gene, cDNA, mRNA, or protein fromany organism and is capable of activating lipoprotein lipase.

Nucleic acid and protein sequences for apoC-II are publicly available.For example, GenBank Accession No. NM_(—)009695 (human) discloses anapoC-II nucleic acid sequence, and GenBank Accession Nos. AAH05348(human), NP_(—)001078821 (rat), NP_(—)001095850 (bovine), andNP_(—)033825 (mouse) disclose apoC-II protein sequences, all of whichare incorporated by reference as provided by GenBank on Mar. 31, 2008.

In one example, apoC-II includes a full-length wild-type (or native)sequence. In other examples, apoC-II includes fragments of a wild-type(or native) sequence. In certain examples, apoC-II fragments include atleast residues 51 to 79, such as 56 to 79, 59 to 79, or 61 to 79 ofapoC-II.

In some examples, apoC-II variants include 19 amino acids with tyrosineat position 3, Ile at position 6, Asp at position 9, and Gln at position10. In some examples, apoC-II has at least 20% sequence identity, forexample, at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%,95%, or 98% sequence identity to human apoC-II residues 59 to 79.

Domain: A domain of a protein is a part of a protein that shares commonstructural, physiochemical and functional features; for examplehydrophobic, polar, globular, helical domains or properties, for examplea DNA binding domain, an ATP binding domain, lipoprotein lipaseactivating domain, and the like. In a particular example, a peptideincludes a first domain and a second domain (though they can occur inany order) in which the first domain is capable of promoting lipidefflux and the second domain is capable of activating lipoproteinlipase. For example, the first domain is an amphipathic α-helical domainwhich has a high hydrophobic moment and the second domain is an apoC-IIprotein, variant, or fragment thereof that is capable of activatinglipoprotein lipase.

Dyslipidemic disorder: A disorder associated with any altered amount ofany or all of the lipids or lipoproteins in the blood. Dyslipidemicdisorders include, for example, hyperlipidemia, hyperlipoproteinemia,hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoA-Ideficiency, and cardiovascular disease (i.e., coronary artery disease,atherosclerosis and restenosis).

Efflux: The process of flowing out. As applied to the results describedherein, lipid efflux refers to a process whereby lipid, such ascholesterol and phospholipid, is complexed with an acceptor, such as anapolipoprotein or apolipoprotein peptide mimic, and removed fromvesicles or cells. “ABCA1-dependent lipid efflux” (or lipid efflux by an“ABCA1-dependent pathway”) refers to a process whereby apolipoproteinsor peptide mimics of apolipoproteins bind to a cell and efflux lipidfrom the cell by a process that is facilitated by the ABCA1 transporter.

Helix: The molecular conformation of a spiral nature, generated byregularly repeating rotations around the backbone bonds of amacromolecule.

Hydrophobic: A hydrophobic (or lipophilic) group is electrically neutraland nonpolar, and thus prefers other neutral and nonpolar solvents ormolecular environments. Examples of hydrophobic molecules includealkanes, oils and fats.

Hydrophobic moment (μ_(H)): One measure of the degree of amphipathicity(i.e., the degree of asymmetry of hydrophobicity) in a peptide or othermolecule; it is the vectorial sum of all the hydrophobicity indices fora peptide, divided by the number of residues. Thus, hydrophobic momentis the hydrophobicity of a peptide measured for different angles ofrotation per amino acid residue. Methods for calculating μ_(H) for aparticular peptide sequence are well-known in the art, and aredescribed, for example, in Eisenberg et al., Faraday Symp. Chem. Soc.17:109-120, 1982; Eisenberg et al., J. Mol. Biol. 179:125-142, 1984; andKyte & Doolittle, J. Mol. Biol., 157: 105-132, 1982. The actual μ_(H)obtained for a particular peptide will depend on the type and totalnumber of amino acid residues composing the peptide.

The amphipathicities of peptides of different lengths can be directlycompared by way of the mean hydrophobic moment. The mean hydrophobicmoment can be obtained by dividing p_(H) by the number of residues inthe helix.

The relative hydrophobic moment (μHrel) of a peptide is its hydrophobicmoment relative to that of a perfectly amphipathic peptide. Thismeasurement provides insight into the amphipathicity using differentscales. For example, a value of 0.5 indicates that the peptide has about50% of the maximum possible amphipathicity. For the CCS scale, therelative hydrophobic moment is calculated by creating a peptide made uponly of Phe (Hi=+10) and Arg (Hi=−10), and placing them so as to obtainperfectly segregated hydrophobic/hydrophilic sectors on the Edmundsonprojection. For the K&D and Eisenberg scales, Ile and Arg are used. Forα-helical peptides (projection angle=100°, an 18 residue peptide with 9Arg, 9 Phe/Ile is used. This results in μHmax of 6.3, 2.8 and 0.83 forthe CCS, K&D and Eisenberg scales, respectively. Table 1 provides thehydrophobicity values for the various amino acid residues using thevarious hydrophobicity scales.

TABLE 1 HYDROPHOBICITY SCALES Residue Symbol CCS Kyte-DoolittleEisenberg Hi Hi Hi Ile I 8.7 4.5 0.73 Leu L 9.7 3.8 0.53 Trp W 9.7 −0.90.37 Phe F 10.0 2.8 0.61 Val V 4.1 4.2 0.54 Met M 4.6 1.9 0.26 Tyr Y 2.5−1.3 0.02 Ala A −1.1 1.8 0.25 Pro P −0.2 −1.6 −0.07 Thr T −3.8 −0.7−0.18 Ser S −4.3 −0.8 −0.26 Cys C −2.3 2.5 0.04 Gly G −2.4 −0.4 0.16 AsnN −7.1 −3.5 −0.64 Asp D −8.3 −3.5 −0.72 Gln Q −6.0 −3.5 −0.69 Glu E −8.3−3.5 −0.62 His H −3.8 −3.2 −0.40 Lys K −9.9 −3.9 −1.10 Arg R −10 −4.5−1.80 Nle Z 9.1 norleucine — Deg 1 13.7 dipropylglycine — Deg 2 6.0diethylglycine — Nva 3 5.3 norvaline — Aib B 1.1 aminoisobutyric acid —Abu U 1.7 aminobutyric acid — Orn O −9 ornithine — Hse 5 −3.5 homoserine— Dap 5 −9.5 diaminopropionic acid — Dab 6 −9.3 diaminobutyric acid —Acp 5 6.2 aminocyclopentanoic acid —

Peptide analysis tool programs (including programs available on theinternal can be used to calculate hydrophobic moment of amphipathicsequences. See, for instance, the peptide sequence analysis tool createdby Alex Tossi and Luca Sandri available on the World Wide Web (www) atbbcm.univ.trieste.it/˜tossi/HydroCalc/HydroMCalc.html (maintained by theUniversity of Trieste, Italy). This tool is also discussed in Tossi etal. (“New Consensus hydrophobicity scale extended to non-proteinogenicamino acids”, PEPTIDES 2002, Proc. of 27^(th) European PeptideSymposium, Sorrento, 2002), incorporated herein by reference. Inparticular, this tool utilizes a Java applet that calculates the meanhydrophobicity, the mean hydrophobic moment and the relative hydrophobicmoment for peptides using a selected scale and a selected projectionangle; this program is known as HydroMCalc.

Ordinary skilled artisans will recognize other ways in which hydrophobicmoment and other comparative measurements of amphipathicity can becalculated.

Hydrophilic: A hydrophilic (or lipophobic) group is electricallypolarized and capable of H-bonding, enabling it to dissolve more readilyin water than in oil or other “non-polar” solvents.

Implant: A support device. For example, an implant is a device that isemployed to enhance and support existing passages, channels, andconduits such as the lumen of a blood vessel. In an example, an implantis an endovascular support. In a particular example, an implant is astent. In one example, an implant is effective to maintain a vesselopen. In the present disclosure, an implant can be coated with orimpregnated with one or more of the disclosed peptides to assist withthe treatment of a dyslipidemic or vascular disorder.

Inhibiting or treating a disease: Inhibiting the full development of adisease, disorder or condition, for example, in a subject who is at riskfor a disease such as atherosclerosis and cardiovascular disease.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease, pathological condition or symptom, refers to any observablebeneficial effect of the treatment. The beneficial effect can beevidenced, for example, by a delayed onset of clinical symptoms of thedisease in a susceptible subject, a reduction in severity of some or allclinical symptoms of the disease, a slower progression of the disease, areduction in the number of relapses of the disease, an improvement inthe overall health or well-being of the subject, or by other parameterswell known in the art that are specific to the particular disease.

Isolated/purified: An “isolated” or “purified” biological component(such as a nucleic acid, peptide or protein) has been substantiallyseparated, produced apart from, or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, that is, other chromosomal and extrachromosomal DNA and RNA, andproteins. Nucleic acids, peptides and proteins that have been “isolated”thus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids, peptides andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids or proteins. The term “isolated” or“purified” does not require absolute purity; rather, it is intended as arelative term. Thus, for example, an isolated biological component isone in which the biological component is more enriched than thebiological component is in its natural environment within a cell.Preferably, a preparation is purified such that the biological componentrepresents at least 50%, such as at least 70%, at least 90%, at least95%, or greater of the total biological component content of thepreparation.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker: A molecule that joins two other molecules, either covalently, orthrough ionic, van der Waals or hydrogen bonds. In particular examples,a linker comprises glycine, proline or alanine. In one specific example,the linker is a sequence of one or more prolines.

Lipid: A class of water-insoluble, or partially water insoluble, oily orgreasy organic substances, that are extractable from cells and tissuesby nonpolar solvents, such as chloroform or ether. Types of lipidsinclude triglycerides (i.e., natural fats and oils composed of glycerinand fatty acid chains), phospholipids (e.g., phosphatidylethanolamine,phosphatidylcholine, phosphatidylserine, and phosphatidylinositol),sphingolipids (e.g., sphingomyelin, cerebrosides and gangliosides), andsterols (e.g., cholesterol).

Lipid affinity: A measurement of the relative binding affinity of anamphipathic α-helix for lipids. Any number of methods well know to oneof skill in the art can be used to determine lipid affinity. In oneembodiment, the lipid affinity of an amphipathic α-helix is determinedby calculating the hydrophobic moment score of the amphipathic α-helix.For example, an amphipathic α-helix with relatively high lipid affinitywill have a hydrophobic moment score per residue greater than or equalto about 0.34 on the Eisenberg scale (100 degree alpha helix), while anamphipathic α-helix with relatively low lipid affinity will have ahydrophobic moment score per residue of less than about 0.34 on theEisenberg scale (Eisenberg et al., Faraday Symp. Chem. Soc. 17:109-120,1982). In an alternative embodiment, an amphipathic α-helix withrelatively high lipid affinity has a hydrophobic moment score perresidue of about 0.40 to about 0.60 on the Eisenberg consensus scale,while a low lipid affinity helix will have a hydrophobic moment scoreper residue of about 0.20 to about 0.40 on the consensus scale(Eisenberg et al., PNAS 81:140-144, 1984 and Eisenberg et al., J. Mol.Biol. 179:125-142, 1984). With any one peptide or peptide analog withmultiple amphipathic α-helical domains, it is to be understood that thedifference between the hydrophobic moment scores of the amphipathicα-helix with the relatively high lipid affinity and the amphipathicα-helix with the relatively low lipid affinity is at least 0.01 on theconsensus scale. In some embodiments, the difference is higher than0.01, such as 0.02, 0.05, 0.08 or 0.1.

In other embodiments, the lipid affinity of an amphipathic α-helix isdetermined by one or more functional tests. Specific, non-limitingexamples of functional tests include: retention time on reverse phaseHPLC, surface monolayer exclusion pressure (Palgunachari et al.,Arterioscler. Thromb. Vasc. Biol. 16:328-338, 1996), binding affinity tophospholipid vesicles (Palgunachari et al., Arterioscler. Thromb. Vasc.Biol. 16:328-338, 1996), and DMPC vesicle solubilization (Remaley etal., J. Lipid Res. 44:828-836, 2003).

Further non-limiting examples of alternative methods of calculating thelipid affinity of an amphipathic α-helix include: total hydrophobicmoment, total peptide hydrophobicity, total peptide hydrophobicity perresidue, hydrophobicity of amino acids on the hydrophobic face, meanrelative hydrophobic moment, hydrophobicity per residue of amino acidson the hydrophobic face, and calculated lipid affinity based onpredicted peptide penetration into phospholipid bilayers (Palgunachariet al., Arterioscler. Thromb. Vasc. Biol. 16:328-338, 1996). Differenttypes of hydrophobicity scales for amino acids also can be used forcalculating hydrophobic moments of amphipathic helices, which can resultin a different relative ranking of their lipid affinity (Kyte et al., J.Mol. Biol. 157:105-132, 1982).

Lipoprotein lipase (LPL): An enzyme that hydrolyzes lipids inlipoproteins, such as those present in chylomicrons and very low-densitylipoproteins (VLDLs), into three free fatty acids and one glycerolmolecule. A deficiency in lipoprotein lipase activity can lead tohypertriglyceridemia. In an example, LPL is activated (e.g., stimulatedor turned-on) by an apoC-II protein, such as a fragment of an apoC-IIprotein including the third helix of apoC-II. For example, LPL isactivated by a polypeptide that includes a domain with an amino acidsequence set forth in any of SEQ ID NOs: 63-69. LPL activation can bemeasured by methods known of those to skill in the art including thosedescribed in detail in Example 10. In an example, LPL activation can bedetermined in vivo by obtaining a sample from a subject and measuringtriglyceride levels in such sample prior to and after treatment with oneor more of the disclosed peptides. A peptide that increases triglyceridelevels above the baseline value by more than 3 times (x's; e.g., 3×) thecoefficient of variation of the assay, which for most triglyceridesassays would be approximately 2-5%, is considered a peptide thatinhibits LPL. Any peptide that lowers the serum triglyceride compared tothe result obtained with the in vivo administration of the 5A peptide bymore than 3× the coefficient of variation of triglyceride assay isconsidered a peptide that activates LPL. A potentially clinically usefulLPL-activating peptide is a peptide that, when administered to subjects,results in a triglyceride level of less than 150 mg/dl, such as lessthan 145 mg/dl, 140 mg/dl, 135 mg/dl, 130 mg/dl, 125 mg/dl or 120 mg/dl.

Non-cytotoxic: A non-cytotoxic compound is one that does notsubstantially affect the viability or growth characteristics of a cellat a dosage normally used to treat the cell or a subject. Furthermore,the percentage of cells releasing intracellular contents, such as LDH orhemoglobin, is low (e.g., about 10% or less) in cells treated with anon-cytotoxic compound. Lipid efflux from a cell that occurs by anon-cytotoxic compound results in the removal of lipid from a cell by aprocess that maintains the overall integrity of the cell membrane anddoes not lead to significant cell toxicity.

Non-polar: A non-polar compound is one that does not have concentrationsof positive or negative electric charge. Non-polar compounds, such as,for example, oil, are not well soluble in water.

Peptide: A polymer in which the monomers are amino acid residues whichare joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “peptide” or “polypeptide” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “peptide” is specificallyintended to cover naturally occurring peptides, as well as those whichare recombinantly or synthetically produced. The term “residue” or“amino acid residue” includes reference to an amino acid that isincorporated into a peptide, polypeptide, or protein.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more multi-domainpeptides or peptide analogs and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Phospholipid: A phospholipid consists of a water-soluble polar head,linked to two water-insoluble non-polar tails (by a negatively chargedphosphate group). Both tails consist of a fatty acid, each about 14 toabout 24 carbon groups long. When placed in an aqueous environment,phospholipids form a bilayer or micelle, where the hydrophobic tailsline up against each other. This forms a membrane with hydrophilic headson both sides. A phospholipid is a lipid that is a primary component ofanimal cell membranes.

Polar: A polar molecule is one in which the centers of positive andnegative charge distribution do not converge. Polar molecules arecharacterized by a dipole moment, which measures their polarity, and aresoluble in other polar compounds and virtually insoluble in nonpolarcompounds.

Recombinant nucleic acid: A sequence that is not naturally occurring orhas a sequence that is made by an artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by chemical synthesis or, more commonly, by theartificial manipulation of isolated segments of nucleic acids, forexample, by genetic engineering techniques such as those described inSambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2^(nd)ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The term recombinant includes nucleic acids that have beenaltered solely by addition, substitution, or deletion of a portion ofthe nucleic acid.

Therapeutically effective amount: A quantity of a specified agent (orcombination of agents) sufficient to achieve a desired effect in asubject being treated with that agent. For example, this can be theamount of a multi-domain peptide or peptide analog useful in preventing,ameliorating, and/or treating a dyslipidemic disorder (e.g.,atherosclerosis) in a subject. Ideally, a therapeutically effectiveamount of an agent is an amount sufficient to prevent, ameliorate,and/or treat a dyslipidemic disorder (e.g., atherosclerosis) in asubject without causing a substantial cytotoxic effect (e.g., membranemicrosolubilization) in the subject. The effective amount of an agentuseful for preventing, ameliorating, and/or treating a dyslipidemicdisorder (e.g., atherosclerosis) in a subject will be dependent on thesubject being treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. In an example, atherapeutically effective amount of one of the disclosed peptides (orpeptide mixtures, such as a peptide including a first domain capable ofpromoting lipid efflux and a second domain having lipoprotein lipaseactivity) results in a triglyceride level of at least less than 150mg/dl, such as less than 145 mg/dl, less than 140 mg/dl, less than 135mg/dl, less than 130 mg/dl, less than 125 mg/dl or less than 120 mg/dl.

Transformed: A “transformed” cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Theterm encompasses all techniques by which a nucleic acid molecule mightbe introduced into such a cell, including transfection with viralvectors, transformation with plasmid vectors, and introduction of nakedDNA by electroporation, lipofection, and particle gun acceleration.

III. Overview of Several Embodiments

Isolated peptides and peptide analogs with multiple amphipathicα-helical domains that promote lipid efflux from cells via anABCA1-dependent pathway are disclosed herein. In one embodiment, themulti-domain peptides include multiple amphipathic α-helical domains,wherein a first amphipathic α-helical domain exhibits higher lipidaffinity compared to a second amphipathic α-helical domain (as measured,e.g., by their hydrophobic moments; see Eisenberg et al., Faraday Symp.Chem. Soc. 17:109-120, 1982; Eisenberg et al., PNAS 81:140-144, 1984;and Eisenberg et al., J. Mol. Biol. 179:125-142, 1984), and wherein thepeptide or peptide analog promotes lipid efflux from cells by anABCA1-dependent pathway.

In specific, non-limiting examples, the first amphipathic α-helicaldomain has a hydrophobic moment score (Eisenberg scale; 100 degree-alphahelix) per residue of about 0.3 to about 0.60 and the second amphipathicα-helical domain has a hydrophobic moment score per residue of about 0.1to about 0.33, wherein the difference between the hydrophobic momentscores of the first amphipathic α-helix and the second amphipathicα-helix is at least 0.01. In some embodiments, the difference is higherthan 0.01, such as 0.02, 0.05, 0.08 or 0.1. For example, the 5A peptide(SEQ ID NO: 3) has a hydrophobic moment score (Eisenberg scale; 100degree-alpha helix) per reside of 0.34 for the N-terminal lipid affinityhelix and a hydrophobic moment score per residue of 0.28 for theC-terminal low lipid affinity helix. Using an alternative scalecalculation, the 5A peptide (SEQ ID NO: 3) has a hydrophobic momentscore 0.4905 for the N-terminal high lipid affinity helix and ahydrophobic moment score per residue of 0.3825 for the C-terminal lowlipid affinity helix. Optionally, the order of relatively high andrelatively low amphipathic helices can be reversed in the peptide.

Using a relative mean hydrophobic moment score, which is normalized to a“perfect” amphipathic helix with a maximum score of 0.83, the twohelices of the 5A peptide (SEQ ID NO: 3) have values of 0.42 and 0.34.It is well recognized that different physical properties, however, canbe used for determining the hydrophobicity of amino acids, which resultsin different scales for calculating the hydrophobic moment of peptides.Calculations with these different scales can change the absolute valueof the hydrophobicity scores and the relative ranking of the lipidaffinity of amphipathic helices. For example, using the Kyte & Doolittlescale (Kyte et al., J. Mol. Biol. 157:105-132, 1982), the N-terminal andC-terminal helices of the 5A peptide would be seen to have hydrophobicmoment scores of 1.47 and 1.26, with a relative mean hydrophobic momentscores of 0.51 and 0.44 (perfect helix: 2.8). Using a combined consensusscale, which is a hybrid of several different scoring systems, theN-terminal and C-terminal helices of the 5A peptide would havehydrophobic moment scores of 4.01 and 2.02, with a relative meanhydrophobic moment score of 0.64 an 0.32 (perfect helix: 6.3). All suchscales, calculations, and measurements can be used, converted andinterchanged, as recognized by those of ordinary skill in the art.

Other representative non-limiting example peptides with multipleamphipathic α-helical domains are shown in SEQ ID NOs: 4-45.

Optionally, the isolated peptides and peptide analogs that promoteABCA1-dependent lipid efflux from cells are also substantiallynon-cytotoxic, for instance, do not significantly increase triglycerides(e.g., triglyceride levels less than 150 mg/dl or do not show astatically significant increase above baseline triglycerides values.Disclosed herein are also isolated peptides and peptide analogs (orcombinations of peptides or analogs, which are not necessarilycovalently linked one to another) capable of promoting lipid efflux fromcells by an ABCA1-dependent pathway and activating lipoprotein lipase.In one embodiment, the isolated peptides and peptide analogs (ormixtures) include a first domain that promotes lipid efflux and a seconddomain that activates lipoprotein lipase; optionally, these domains arecovalently linked, with or without the presence of a linking peptide orother bridging molecule.

In specific non-limiting examples, the first domain is an amphipathicα-helical domain with a hydrophobic moment score (Eisenberg scale; 100degree-alpha helix) per residue of about 0.34 to about 0.60 and thesecond domain is an amphipathic α-helical domain of apoC-II protein or avariant or fragment thereof that retains a lipoprotein lipase-activatingproperty, such as a domain comprising an amino acid sequence set forthin any one of SEQ ID NOs: 63-69. Representative non-limiting examplepeptides with both lipid efflux promoting activity and lipoproteinlipase activating activity are shown in SEQ ID NOs: 70-75. In oneexample, a peptide has the amino acid sequence set forth in SEQ ID NO:70. LPL activation can be measured by methods known of those to skill inthe art including those described in detail in Example 10. In anexample, LPL activation can be determined in vivo by obtaining a samplefrom a subject and measuring triglyceride levels in such sample prior toand after treatment with one or more of the disclosed peptides. Atriglyceride level of less than 150 mg/dl following treatment of asubject (such as a human subject) with a test compound indicates thatthe compound (e.g., apoC-II protein, fragment or variant thereof) is aLPL activator.

The isolated peptides and peptide analogs disclosed herein can alsoinclude an additional functional domain or peptide. Specific,non-limiting examples of the additional functional domains or peptidesinclude a heparin binding site, an integrin binding site, a P-selectinsite, a TAT HIV sequence, a panning sequence, a penatratin sequence, aSAA C-terminus sequence, a SAA N-terminus sequence, a LDL receptorsequence, a modified 18A sequence, an apoA-I Milano sequence, a 6×-Hissequence, a lactoferrin sequence, a lipoprotein lipase activating domain(such as a fragment or variant of apoC-II) or combinations of two ormore thereof.

Pharmaceutical compositions are disclosed that include one or moreisolated peptides or peptide analogs with multiple amphipathic α-helicaldomains that promote lipid efflux from cells via an ABCA1-dependentpathway. Representative peptides with multiple amphipathic α-helicaldomains are shown in SEQ ID NOs: 3-45. Pharmaceutical compositions arealso disclosed that include one or more isolated peptides or peptideanalogs that promote lipid efflux from cells via an ABCA1-dependentpathway and activate lipoprotein lipase, such as representative peptidesshown in SEQ ID NOs: 70-75. In one specific example, the pharmaceuticalcomposition includes a peptide with the amino acid sequence of SEQ IDNO: 70.

Methods are provided for treating or inhibiting dyslipidemic andvascular disorders in a subject. In one embodiment, this method includesadministering to the subject a therapeutically effective amount of apharmaceutical composition that includes one or more isolated peptidesor peptide analogs with two or more amphipathic α-helical domains thatpromote lipid efflux from cells via an ABCA1-dependent pathway andactivate lipoprotein lipase. In specific, non-limiting examples, thedyslipidemic and vascular disorders include hyperlipidemia,hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDLdeficiency, apoA-I deficiency, coronary artery disease, atherosclerosis,thrombotic stroke, peripheral vascular disease, restenosis, acutecoronary syndrome, and reperfusion myocardial injury.

In certain examples of the provided method, the isolated peptideincludes two amphipathic α-helical domains in which the first domain haslipid efflux promoting activity and the second domain has lipoproteinlipase activity (such as an apoC-II protein, fragment or variant with anamino acid sequence as set forth in SEQ ID NOs: 63-69). In one example,the isolated peptide includes the amino sequence of SEQ ID NO: 70. Inother examples of the provided method, the isolated peptide includes twoor more amphipathic α-helical domain capable of promoting lipid effluxwith an apoC-II variant or fragment (such as provided in SEQ ID NO: 69)attached to the C-terminal end of at least one of the lipid effluxpromoting amphipathic α-helical domains.

In other non-limiting examples of the provided method, the methodincludes administering to the subject a therapeutically effective amountof a pharmaceutical composition that includes at least one isolatedpeptide (or peptide analog) with multiple amphipathic α-helical domainsand that promotes lipid efflux from cells via an ABCA1-dependent pathway(such as an isolated peptide that includes two amphipathic α-helicaldomains and has an amino acid sequence as set forth in SEQ ID NOs: 3-45)and a separate lipoprotein lipase activating agent, such as an apoC-IIprotein, variant or fragment thereof (e.g., peptide) that retainslipoprotein lipase activity (e.g., results in triglyceride levels ofless than 150 mg/dl). The peptide that promotes efflux need not becovalently linked to the peptide that has LPL activating activity.

Methods for identifying non-cytotoxic peptides that promoteABCA1-dependent lipid efflux from cells and/or peptides that activatelipoprotein lipase in cells (or peptides that have both activities) arealso disclosed.

IV. Multi-Domain Amphipathic Peptides

Isolated peptides and peptide analogs including peptides with multipleamphipathic α-helical domains that promote lipid efflux from cells viaan ABCA1-dependent pathway have been identified and are described in,for instance, PCT Publication No. WO 2006/044596 (PCT/US2005/036933), aswell as the corresponding U.S, National Stage application (U.S. patentapplication Ser. No. 11/577,259), which are incorporated herein byreference in their entirety. Various peptides described therein includeda first amphipathic α-helical domain exhibited higher lipid affinityrelative to a second amphipathic α-helical domain, in the same peptide.

ApoA-I, the predominant protein constituent of HDL (Panagotopulos etal., J. Biol. Chem. 277:39477-39484, 2002), is believed to promote lipidefflux from cells by a detergent-like extraction process (Remaley etal., J. Lipid Res. 44:828-836, 2003). The ABCA1 transporter has beenproposed to facilitate this process by creating a lipid microdomain thatpromotes the binding of apoA-I to cells and creates a lipid domain thatis susceptible for removal by apoA-I by a detergent-like extractionprocess. ApoA-I, like most of the other natural exchangeable typeapolipoproteins, is almost completely dependent upon the presence ofABCA1 for promoting lipid efflux (Remaley et al., Biochem. Biophys. Res.Commun. 280:818-823, 2001). Furthermore, when lipid efflux occurs byapoA-I and the other natural exchangeable type apolipoproteins, itoccurs by a non-cytotoxic process, whereby the integrity of the cellmembrane is maintained (Remaley et al., J. Lipid Res. 44:828-836, 2003).ApoA-I contains at least 8 large amphipathic helical domains, which havea wide range of lipid affinity (Gillote et al., J. Biol. Chem.274:2021-2028, 1999).

Synthetic peptides of each helix of apoA-I have been made, and it hasbeen shown that only 2 of the 8 large amphipathic helices of apoA-I,which have relatively high lipid affinity, can by themselves promotelipid efflux from cells in culture (Gillote et al., J. Biol. Chem.274:2021-2028, 1999 and Palgunachari et al., Arterioscler. Thromb. Vasc.Biol. 16:328-338, 1996). Additionally, synthetic peptide mimics ofapolipoproteins have been shown to have anti-inflammatory andanti-oxidant properties (Van Lenten et al., Trends Cardiovasc. Med.11:155-161, 2001; Navab et al., Cur. Opin. Lipidol. 9:449-456, 1998;Barter et al., Cur. Opin. Lipidol. 13:285-288, 2002).

Previously, synthetic peptide mimics of apolipoproteins have beendesigned to have high lipid affinity (Remaley et al., J. Lipid Res.44:828-836, 2003; Segrest et al., J. Lipid Res. 33:141-166, 1992;Anantharamaiah et al., J. Biol. Chem. 260:10248-10255, 1985; Garber etal, J. Lipid Res. 42:545-552, 2001; Navab et al., Circulation105:290-292, 2002; and U.S. Pat. No. 6,156,727), because high lipidaffinity has been shown to be a necessary feature for a peptide tomediate lipid efflux by the ABCA1 transporter (Remaley et al., J. LipidRes. 44:828-836, 2003). It has also been shown, however, that peptidemimics of apoA-I with high lipid affinity can also promote lipid effluxindependent of the ABCA1 transporter (Remaley et al., J. Lipid Res.44:828-836, 2003). Such peptides have been shown to promote lipid effluxfrom cells not expressing the ABCA1 transporter, and from Tangierdisease cells that do not contain a functional ABCA1 transporter(Remaley et al., J. Lipid Res. 44:828-836, 2003). Furthermore, syntheticpeptide mimics of apoA-I that posses high lipid affinity can alsoextract lipid by a passive physical process, based on their ability toremove lipid from cells that have been fixed with paraformaldehyde(Remaley et al., J. Lipid Res. 44:828-836, 2003). Lipid efflux fromcells by this ABCA1-independent pathway has been shown to be cytotoxicto cells, based on the cellular release of LDH (Remaley et al., J. LipidRes. 44:828-836, 2003).

In addition to the undesirable cytotoxic effect on cells,ABCA1-independent lipid efflux may also reduce the therapeutic benefitof such peptides by reducing their in vivo capacity for removing lipidfrom cells affected by the atherosclerotic process. For example, even insubjects with dyslipidemic and vascular disorders, most cells do nothave excess cellular cholesterol and, therefore, do not express theABCA1 transporter. Cells, such as macrophages, endothelial cells andsmooth muscle cells, which are present in atherosclerotic plaques, areall prone to lipid accumulation, and express ABCA1 when loaded withexcess cholesterol. The expression of ABCA1 by these cells has beenshown to be exquisitely regulated by the cholesterol content of cells(Langmann et al., Biochem. Biophys. Res. Commun. 257:29-33, 1999).Induction of the ABCA1 transporter by intracellular cholesterol is aprotective cellular mechanism against excess intracellular cholesteroland has been shown to be critical in preventing the development ofatherosclerosis (Dean and Chimini, J. Lipid Res. 42:1007-1017, 2001).Peptide mimics of apolipoproteins that are not specific for removingcholesterol by the ABCA1 transporter would be less therapeuticallyeffective in removing cholesterol from ABCA1 expressing cells becauseany cholesterol removed by the peptides from the more abundant non-ABCA1expressing cells will reduce the overall total cholesterol bindingcapacity of these peptides. The selective and non-cytotoxic removal oflipid from only cells that express the ABCA1 transporter would,therefore, be a desirable property for therapeutic peptide mimics ofapolipoproteins.

The current disclosure provides isolated multi-domain peptides orpeptide analogs that specifically efflux lipids from cells by the ABCA1transporter in a non-cytotoxic manner. In one embodiment, such peptidesor peptide analogs contain an amphipathic α-helical domain that exhibitsrelatively high lipid affinity (e.g., a hydrophobic moment score(Eisenberg scale; 100 degree-alpha helix) per residue of about 0.3 toabout 0.60) and a second amphipathic α-helical domain with relativelylow lipid affinity (e.g., a hydrophobic moment score per residue ofabout 0.1 to about 0.33), wherein the difference between the hydrophobicmoment scores of the amphipathic α-helix with the relatively high lipidaffinity and the amphipathic α-helix with the relatively low lipidaffinity is at least 0.01. In some embodiments, the difference is higherthan 0.01, such as 0.02, 0.05, 0.08 or 0.10.

Peptides containing one amphipathic α-helix with a relatively high lipidaffinity, when coupled to another α-helix with a relatively low lipidaffinity, are specific for removing lipids from cells by the ABCA1transporter. In particular examples, these peptides are not onlyspecific for removing lipids from cells by the ABCA1 transporter, butare also capable of activating lipoprotein lipase.

The degree of amphipathicity (i.e., degree of asymmetry ofhydrophobicity) in the multi-domain peptides or peptide analogs can beconveniently quantified by calculating the hydrophobic moment (μ_(H)) ofeach of the amphipathic α-helical domains. Methods for calculating μ_(H)for a particular peptide sequence are well-known in the art, and aredescribed, for example in Eisenberg et al., Faraday Symp. Chem. Soc.17:109-120, 1982; Eisenberg et al., PNAS 81:140-144, 1984; and Eisenberget al., J. Mol. Biol. 179:125-142, 1984. The actual μ_(H) obtained for aparticular peptide sequence will depend on the total number of aminoacid residues composing the peptide. The amphipathicities of peptides ofdifferent lengths can be directly compared by way of the meanhydrophobic moment. The mean hydrophobic moment per residue can beobtained by dividing μ_(H) by the number of residues in the peptide.

In another embodiment, such peptides or peptide analogs contain anamphipathic α-helical domain that exhibits relatively high lipidaffinity (e.g., a hydrophobic moment score (Eisenberg scale; 100degree-alpha helix) per residue of about 0.30 to about 0.60) and asecond amphipathic α-helical domain with moderate lipid affinity (e.g.,a hydrophobic moment score (Eisenberg scale; 100 degree-alpha helix) perresidue of about 0.29 to about 0.33), wherein the difference between thehydrophobic moment scores of the amphipathic α-helix with the relativelyhigh lipid affinity and the amphipathic α-helix with the relativelymoderate lipid affinity is at least 0.01. In some embodiments, thedifference is higher than 0.01, such as 0.02, 0.05, 0.08 or 0.1. Suchpeptides have reduced specificity for the ABCA1 transporter, as comparedto peptides containing one amphipathic α-helix with a relatively highlipid affinity and one amphipathic α-helix with a relatively low lipidaffinity, but are still less cytotoxic to cells than peptides thatcontain two amphipathic α-helical domains with relatively high lipidaffinity.

Specific, non-limiting examples of multi-domain peptides with multipleamphipathic α-helical domains that mediate ABCA1-dependent cholesterolefflux from cells are shown in Table 2.

TABLE 2 Exemplary multi-domain peptides that mediate ABCA1-dependent cholesterol efflux from cells. SEQ ID Peptide SequenceNO: 5A-37pA DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA 3 1A-37pADWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKAKEAF 4 2A-37pADWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKAKEAA 5 3A-37pADWLKAFYDKVAEKLKEAFPDWLKAAYDKVAEKAKEAA 6 4A-37pADWLKAFYDKVAEKLKEAFPDWLKAAYDKAAEKAKEAA 7 Pep1DWLKAFYDKVAEKLKEAFPDWGKAGYDKGAEKGKEAG 8 Pep2DWLKAFYDKVAEKLKEAFPDWGKAGYDKGAEKGKEAF 9 Pep3DWGKAGYDKGAEKGKEAGDWLKAFYDKVAEKLKEAF 10 Pep4DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLK 11 Pep5KAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 12 Pep6DWLKAFYDKVAEKLKEAFPDWLKAFYDKVA 13 Pep7 DKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 14Pep8 DWLKAFYDKVAEKLKEAFPDWLKAFYKVAEKLKEAF 15 Pep9DWLKAFYDKVAEKLKEAFPDWLKAFYVAEKLKEAF 16 Pep10DWLAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 17 Pep11DWLFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 18 Pep12DWLKAFYDKVAEKLKEAFPDWLAKAFYDKVAEKLKEAF 19 Pep13DWLKAFYDKVAEKLKEAFPDWLAAKAFYDKVAEKLKEAF 20 Pep14DWLKAAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 21 Pep15DWLKAAAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 22 Pep16DWLKAFYDKVAEKLKEAFPDWLEAFYDKVAKKLKEAF 23 Pep17DWLKAFYDKVAEKLKEAFPDWLEAFYDEVAKKLKKAF 24 Pep18DWLEAFYDKVAKKLKEAFPDWLKAFYDKVAEKLKEAF 25 Pep19DWLEAFYDEVAKKLKKAFPDWLKAFYDKVAEKLKEAF 26 Pep20DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 27 Pep21DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 28 Pep22DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 29 Pep23DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF 30 Pep24LLDNWDSVTSTFSKLREQPDWAKAAYDKAAEKAKEAA 31 Pep25LESFKVSFLSALEEYTKKPDWAKAAYDKAAEKAKEAA 32 Pep26DWAKAAYDKAAEKAKEAAPLLDNWDSVTSTFSKLREQ 33 Pep27DWAKAAYDKAAEKAKEAAPLESFKVSFLSALEEYTKK 34 Pep28DWLKAFYDKVAEKLKEAFPSDELRQRLAARLEALKEN 35 Pep29DWLKAFYDKVAEKLKEAFPRAELQEGARQKLHELQEK 36 Pep30SDELRQRLAARLEALKENPDWLKAFYDKVAEKLKEAF 37 Pep31RAELQEGARQKLHELQEKPDWLKAFYDKVAEKLKEAF 38 Pep32LLDNWDSVTSTFSKLREQPSDELRQRLAARLEALKEN 39 Pep33LESFKVSFLSALEEYTKKPRAELQEGARQKLHELQEK 40 Pep34SDELRQRLAARLEALKENPLLDNWDSVTSTFSKLREQ 41 Pep35LLDNWDSVTSTFSKLREQPLESFKVSFLSALEEYTKK  42 Pep36DWLKAFYDKVAEKLKEAFPDWLRAFYDKVAEKLKEAF 43 Pep37DWLKAFYDKVAEKLKEAFPDWLRAFYDRVAEKLKEAF 44 Pep38DWLKAFYDKVAEKLKEAFPDWLRAFYDRVAEKLREAF 45

Specific, non-limiting examples of α-helical domains capable ofactivating lipoprotein lipase are shown in Table 3.

TABLE 3 Exemplary domains that activate lipoprotein lipase  in cells.SEQ ID Domain Sequence NO: apoC-II DLYSKSTAAMSTYTGIFTDQVLSVLKGEE 63(residue 51-79) apoC-II STAAMSTYTGIFTDQVLSVLKGEE 64 (residue 56-79)apoC-II AMSTYTGIFTDQVLSVLKGEE 65 (residue 59-79) apoC-IISTYTGIFTDQVLSVLKGEE 66 (residue 61-79) Consensus apoC-xxYxxIxxDQxxxxxxxxx; wherein  67 II x is any amino acid that will (residue 61-79) form an amphipathic helix,  will conserve the relativeposition of these 4 residues with respect to theirposition in a helical wheel  plot and allow the domain to activate lipoprotein lipase. Consensus apoC-AxxTYxGIxxDQVLSVLKGEE; wherein  68 II x is any amino acid that will (residue 59-79) form an amphipathic helix, will conserve the relativeposition of these 4 residues with respect to theirposition in a helical wheel plot and allow the domain to activate lipoprotein lipase.

Specific, non-limiting examples of multi-domain peptides with multipleamphipathic α-helical domains capable of mediating ABCA1-dependentcholesterol efflux from cells and activating lipoprotein lipase areshown in Table 4.

TABLE 4 Exemplary multi-domain peptides that mediate ABCA1-dependentcholesterol efflux from cells and are capable of activating lipoprotein lipase.SEQ ID Peptide Sequence NO: 5A-P-apoC-IIDWLKAFYDKVAEKLKEAFPDLYSKSTAAMSTYTGIFTDQVLSV 70 (residue 51- LKGEE 79);(using DLYSKSTAAMSTYTGIFTDQVLSVLKGEE-mean Referred to ashydrophobic moment: CCS scale: 1.86 μH; Kyte & Doolittle: 0.41 μH;18A-C-IIand Eisenberg: 0.17 μH; mean hydrophobicity: CCS scale: −1.13 H;Kyte & Doolittle: −0.36 H; Eisenberg: −0.12 H; mean relativehydrophobic moment: CCS scale: 0.29 μHrel; Kyte & Doolittle: 0.14μHrel; Eisenberg: 0.21 μHrel) 5A-P-apoC-IIDWLKAFYDKVAEKLKEAFPSTAAMSTYTGIFTDQVLSVLKGEE 71 (residue 56-79)(using STAAMSTYTGIETDQVLSVLKGEE-mean hydrophobicmoment: CCS scale: 1.3 μH; Kyte & Doolittle: 0.21 μH; Eisenberg:0.12 μH; mean hydrophobicity: CCS scale: −0.94 H; Kyte & Doolittle:-0.18 H; Eisenberg: −0.05 H; mean relative hydrophobic moment: CCSscale: 0.2 μHrel; Kyte & Doolittle: 0.07 μHrel; Eisenberg: 0.15 μHrel)5A-P-apoC-II DWLKAFYDKVAEKLKEAFPAMSTYTGIFTDQVLSVLKGEE 72 (residue 59-79)(using AMSTYTGIFTDQVLSVLKGEE-mean hydrophobic moment:CCS scale: 1.66 μH; Kyte & Doolittle: 0.37 μH; Eisenberg: 0.16 μH;mean hydrophobicity: CCS scale: −0.63 H; Kyte & Doolittle: −0.22 H;Eisenberg: −0.05 H; mean relative hydrophobic moment: CCS scale:0.26 μHrel; Kyte & Doolittle: 0.12 μHrel; Eisenberg: 0.2 μHrel)5A-P-apoC-II DWLKAFYDKVAEKLKEAFPSTYTGIFTDQVLSVLKGEE 73 (residue 61-79)(using STYTGIFTDQVLSVLKGEE-mean hydrophobic moment:CCS scale: 1.61 μH; Kyte & Doolittle: 0.31 μH; Eisenberg: 0.16 μH;mean hydrophobicity: CCS scale: −0.88 H; Kyte & Doolittle: −0.43 H;Eisenberg: −0.08 H; mean relative hydrophobic moment: CCS scale:0.25 μHrel; Kyte & Doolittle: 0.11 μHrel; Eisenberg: 0.2 μHrel)5A-P-apoC-II DWLKAFYDKVAEKLKEAFPxxYxxIxxDQxxxxxxxxx 74 (residue 61-79)5A-apoC-II DWLKAFYDKVAEKLKEAFPAxxTYxGIxxDQVLSVLKGEE 75 (residue 59-79)

In the multi-domain peptides disclosed herein, the linkage between aminoacid residues can be a peptide bond or amide linkage (i.e., —C—C(O)NH—).Alternatively, one or more amide linkages are optionally replaced with alinkage other than amide, for example, a substituted amide. Substitutedamides generally include, but are not limited to, groups of the formula—C(O)NR—, where R is (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁-C₆)alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted(C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆)alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl,substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl, andsubstituted 6-26 membered alkheteroaryl. Additionally, one or more amidelinkages can be replaced with peptidomimetic or amide mimetic moietieswhich do not significantly interfere with the structure or activity ofthe peptides. Suitable amide mimetic moieties are described, forexample, in Olson et al., J. Med. Chem. 36:3039-3049, 1993.

Additionally, in representative multi-domain peptides disclosed herein,the amino- and carboxy-terminal ends can be modified by conjugation withvarious functional groups. Neutralization of the terminal charge ofsynthetic peptide mimics of apolipoproteins has been shown to increasetheir lipid affinity (Yancey et al., Biochem. 34:7955-7965, 1995;Venkatachalapathi et al., Protein: Structure, Function and Genetics15:349-359, 1993). For example, acetylation of the amino terminal end ofamphipathic peptides increases the lipid affinity of the peptide (Mishraet al., J. Biol. Chem. 269:7185-7191, 1994). Other possible endmodifications are described, for example, in Brouillette et al.,Biochem. Biophys. Acta 1256:103-129, 1995: Mishra et al., J. Biol. Chem.269:7185-7191, 1994; and Mishra et al., J. Biol. Chem. 270:1602-1611,1995.

Furthermore, in representative multi-domain peptides disclosed herein,the amino acid proline is used to link the multiple amphipathicα-helices. However, other suitable amino acids (such as glycine, serine,threonine, and alanine) that functionally separate the multipleamphipathic α-helical domains can be used. In some embodiments, thelinking amino acid will have the ability to impart a β-turn at thelinkage, such as glycine, serine, threonine, and alanine. In addition,larger linkers containing two or more amino acids or bifunctionalorganic compounds, such as H₂N(CH₂)_(n)COOH, where n is an integer from1 to 12, can also be used. Examples of such linkers, as well as methodsof making such linkers and peptides incorporating such linkers, arewell-known in the art (see, e.g., Hunig et al., Chem. Ber.100:3039-3044, 1974 and Basak et al., Bioconjug. Chem. 5:301-305, 1994).

Also encompassed by the present disclosure are modified forms of themulti-domain peptides, wherein one or more amino acids in the peptidesare substituted with another amino acid residue. The simplestmodifications involve the substitution of one or more amino acids foramino acids having similar physiochemical and/or structural properties.These so-called conservative substitutions are likely to have minimalimpact on the activity and/or structure of the resultant peptide.Examples of conservative substitutions are shown below.

Original Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepeptide backbone in the area of the substitution, for example, as ahelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Amino acids are typically classified in one or more categories,including polar, hydrophobic, acidic, basic and aromatic, according totheir side chains. Examples of polar amino acids include those havingside chain functional groups such as hydroxyl, sulfhydryl, and amide, aswell as the acidic and basic amino acids. Polar amino acids include,without limitation, asparagine, cysteine, glutamine, histidine,selenocysteine, serine, threonine, tryptophan and tyrosine. Examples ofhydrophobic or non-polar amino acids include those residues havingnon-polar aliphatic side chains, such as, without limitation, leucine,isoleucine, valine, glycine, alanine, proline, methionine andphenylalanine. Examples of basic amino acid residues include thosehaving a basic side chain, such as an amino or guanidino group. Basicamino acid residues include, without limitation, arginine, homolysineand lysine. Examples of acidic amino acid residues include those havingan acidic side chain functional group, such as a carboxy group. Acidicamino acid residues include, without limitation aspartic acid andglutamic acid. Aromatic amino acids include those having an aromaticside chain group. Examples of aromatic amino acids include, withoutlimitation, biphenylalanine, histidine, 2-napthylalananine,pentafluorophenylalanine, phenylalanine, tryptophan and tyrosine. It isnoted that some amino acids are classified in more than one group, forexample, histidine, tryptophan and tyrosine are classified as both polarand aromatic amino acids. Additional amino acids that are classified ineach of the above groups are known to those of ordinary skill in theart.

The substitutions which in general are expected to produce the greatestchanges in peptide properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

As the lipid affinity of an amphipathic helix is largely due to thehydrophobicity of the amino acid residues on the hydrophobic face of thehelix (Eisenberg et al., PNAS 81:140-144, 1984 and Eisenberg et al., J.Mol. Biol. 179:125-142, 1984), the overall lipid affinity of anamphipathic helix can be reduced by replacing hydrophobic amino acidswith more polar amino acids. In one embodiment, hydrophobic amino acidson the hydrophobic face of the 37-pA peptide (e.g., Phe, Leu or Val)were replaced with Ala, which is less hydrophobic than Phe, Leu and Val(Eisenberg et al., PNAS 81:140-144, 1984 and Eisenberg et al., J. Mol.Biol. 179:125-142, 1984). Specific, non-limiting examples include the5A-37pA peptide (SEQ ID NO: 3), the 1A-37pA peptide (SEQ ID NO: 4), the2A-37pA peptide (SEQ ID NO: 5), the 3A-37pA peptide (SEQ ID NO: 6), andthe 4A-37pA peptide (SEQ ID NO: 7).

In another embodiment, hydrophobic amino acids on the hydrophobic faceof the 37-pA peptide (e.g., Phe, Leu or Val) can be replaced with Gly,which is less hydrophobic than Phe, Leu and Val (Eisenberg et al., PNAS81:140-144, 1984 and Eisenberg et al., J. Mol. Biol. 179:125-142, 1984).Specific, non-limiting examples include those peptides shown in SEQ IDNOs: 8-10. Other slightly hydrophobic amino acids can be used in placeof Ala or Gly for the substitutions (Eisenberg et al., PNAS 81:140-144,1984 and Eisenberg et al., J. Mol. Biol. 179:125-142, 1984).

In addition to the naturally occurring genetically encoded amino acids,amino acid residues in the multi-domain peptides may be substituted withnaturally occurring non-encoded amino acids and synthetic amino acids.Certain commonly encountered amino acids which provide usefulsubstitutions include, but are not limited to, β-alanine and otheromega-amino acids, such as 3-aminopropionic acid, 2,3-diaminopropionicacid, 4-aminobutyric acid and the like; α-aminoisobutyric acid;ε-aminohexanoic acid; δ-aminovaleric acid; N-methylglycine or sarcosine;ornithine; citrulline; t-butylalanine; t-butylglycine;N-methylisoleucine; phenylglycine; cyclohexylalanine; norleucine;naphthylalanine; 4-chlorophenylalanine; 2-fluorophenylalanine;3-fluorophenylalanine; 4-fluorophenylalanine; penicillamine;1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; 3-2-thienylalanine;methionine sulfoxide; homoarginine; N-acetyl lysine; 2,4-diaminobutyricacid; 2,3-diaminobutyric acid; p-aminophenylalanine; N-methyl valine;homocysteine; homophenylalanine; homoserine; hydroxyproline;homoproline; N-methylated amino acids; and peptoids (N-substitutedglycines).

While in certain embodiments, the amino acids of the multi-domainpeptides will be substituted with L-amino acids, the substitutions arenot limited to L-amino acids. Thus, also encompassed by the presentdisclosure are modified forms of the multi-domain peptides, wherein anL-amino acid is replaced with an identical D-amino acid (e.g.,L-Arg→D-Arg) or with a conservatively-substituted D-amino acid (e.g.,L-Arg→D-Lys), and vice versa. Specific, non-limiting examples includethose peptides shown in SEQ ID NOs: 27-30 (see Table 2; substitutedamino acids are underlined).

In addition to making amino acid substitutions, other methods can beused to reduce the lipid affinity of an amphipathic α-helical domain.Examples of such methods include shortening the helical domain(specific, non-limiting examples include those peptides shown in SEQ IDNOs: 11-14), adding or deleting one or more amino acids to change thehelix's phase (specific, non-limiting examples include those peptidesshown in SEQ ID NOs: 19-22 and 15-18, respectively), and changing theType A amphipathic helical charge distribution of the polar face byswitching the location of the positive and negative charge residues(specific, non-limiting examples include those peptides shown in SEQ IDNOs: 23-26; Segrest et al., Adv. Protein Chem. 45:303-369, 1994).Additional methods include, for example, combining natural high lipidaffinity helices with artificially designed low lipid affinity helices(specific, non-limiting examples include those peptides shown in SEQ IDNOs: 31-34), combining natural low lipid affinity helices withartificially designed high lipid affinity helices (specific,non-limiting examples include those peptides shown in SEQ ID NOs:35-38), and combining non-contiguous natural low lipid affinity heliceswith natural high lipid affinity helices (specific, non-limitingexamples include those peptides shown in SEQ ID NOs: 39-42). ReplacingLys residues at the interface between the hydrophobic and hydrophilicface with Arg (which decreases the ability of amphipathic peptides toinsert in phospholipid bilayers, Palgunachari et al., Arterioscler.Thromb. Vasc. Biol. 16:328-338, 1996), is an additional method ofreducing the lipid affinity of an amphipathic α-helical domain(specific, non-limiting examples include those peptides shown in SEQ IDNOs: 43-45).

Many of these changes to the amphipathic helix will be reflected in adecrease in the hydrophobic moment of the peptide. However, somemodifications (e.g., D-amino acid substitutions, changes to the chargedistribution of the polar face residues and replacing Lys residues withArg residues) of the amphipathic helix may not alter the calculatedhydrophobic moment, but will reduce the lipid affinity of the peptide.In such instances, a functional test of lipid affinity, such asretention time on reverse phase HPLC can be used to assess the impact ofany change on the lipid affinity of the peptide (see, e.g., FIG. 8).Additional, non-limiting examples of functional tests that can be usedto measure the lipid affinity of the multi-domain peptides disclosedherein include: surface monolayer exclusion pressure (Palgunachari etal., Arterioscler. Thromb. Vasc. Biol. 16:328-338, 1996), bindingaffinity to phospholipid vesicles (Palgunachari et al., Arterioscler.Thromb. Vasc. Biol. 16:328-338, 1996) and DMPC vesicle solubilization(Remaley et al., J. Lipid Res. 44:828-836, 2003). Further examples ofalternative methods of calculating the predicted lipid affinity of themulti-domain peptides include: total hydrophobic moment, total peptidehydrophobicity, total peptide hydrophobicity per residue, hydrophobicityof amino acids on the hydrophobic face, hydrophobicity per residue ofamino acids on the hydrophobic face, and calculated lipid affinity basedon predicted peptide penetration into phospholipid bilayers(Palgunachari et al., Arterioscler. Thromb. Vasc. Biol. 16:328-338,1996). Regardless of the parameter(s) used to assess the lipid affinityof the multi-domain peptides, those peptides that contain at least twoor more helices, with at least one helix having relatively high lipidaffinity and one helix having relatively low lipid affinity, areconsidered to be encompassed by the present disclosure. If alternativetests or alternative calculations are used instead of the hydrophobicmoment calculation for calculating lipid affinity, the optimal value oflipid affinity for the high and low lipid affinity helices can befunctionally determined by performing cytotoxicity assays (see, e.g.,FIG. 9) and lipid efflux assays on non-ABCA1 expressing and ABCA1expressing cells (see, e.g., FIG. 10).

Also encompassed by the present disclosure are multi-domain peptides orpeptide analogs, wherein the multiple amphipathic α-helical domains arecomprised of dimers, trimers, tetramers and even higher order polymers(i.e., “multimers”) comprising the same or different sequences. Suchmultimers may be in the form of tandem repeats. The amphipathicα-helical domains may be directly attached to one another or separatedby one or more linkers. The amphipathic α-helical domains can beconnected in a head-to-tail fashion (i.e., N-terminus to C-terminus), ahead-to-head fashion, (i.e., N-terminus to N-terminus), a tail-to-tailfashion (i.e., C-terminus to C-terminus), and/or combinations thereof.In one embodiment, the multimers are tandem repeats of two, three, four,and up to about ten amphipathic α-helical domains, but any number ofamphipathic α-helical domains that has the desired effect ofspecifically promoting ABCA1 lipid efflux with low cytotoxicity can beused.

Additional aspects of the disclosure include analogs, variants,derivatives, and mimetics based on the amino acid sequence of themulti-domain peptides disclosed herein. Typically, mimetic compounds aresynthetic compounds having a three-dimensional structure (of at leastpart of the mimetic compound) that mimics, for example, the primary,secondary, and/or tertiary structural, and/or electrochemicalcharacteristics of a selected peptide, structural domain, active site,or binding region (e.g., a homotypic or heterotypic binding site, acatalytic active site or domain, a receptor or ligand binding interfaceor domain, or a structural motif) thereof. The mimetic compound willoften share a desired biological activity with a native peptide, asdiscussed herein (e.g., the ability to interact with lipids). Typically,at least one subject biological activity of the mimetic compound is notsubstantially reduced in comparison to, and is often the same as orgreater than, the activity of the native peptide on which the mimeticwas modeled.

A variety of techniques well known to one of skill in the art areavailable for constructing peptide mimetics with the same, similar,increased, or reduced biological activity as the corresponding nativepeptide. Often these analogs, variants, derivatives and mimetics willexhibit one or more desired activities that are distinct or improvedfrom the corresponding native peptide, for example, improvedcharacteristics of solubility, stability, lipid interaction,susceptibility to hydrolysis or proteolysis (see, e.g., Morgan andGainor, Ann. Rep. Med. Chem. 24:243-252, 1989) and/or lipoprotein lipaseactivity. For example, a peptide mimetic with lipoprotein lipaseactivity may be generated by incorporating apoC-II amino acid residuesknown to be involved in apoC-II lipoprotein lipase activation (such asone or more of the amino acids provided in SEQ ID NO:69) into one of thedisclosed multi-domain peptides. In one particular example, a peptidemimetic with lipid efflux activity and lipoprotein lipase activatingactivity is a peptide with an amino acid sequence provided by SEQ IDNOs: 70-75, such as SEQ ID NO: 70.

In addition, mimetic compounds of the disclosure can have other desiredcharacteristics that enhance their therapeutic application, such asincreased cell permeability, greater affinity and/or avidity for abinding partner, and/or prolonged biological half-life. The mimeticcompounds of the disclosure can have a backbone that is partially orcompletely non-peptide, but with side groups identical to the sidegroups of the amino acid residues that occur in the peptide on which themimetic compound is modeled. Several types of chemical bonds, forexample, ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant mimetic compounds.

In one embodiment, multi-domain peptides useful within the disclosureare modified to produce peptide mimetics by replacement of one or morenaturally occurring side chains of the 20 genetically encoded aminoacids (or D-amino acids) with other side chains, for example with groupssuch as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl,amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, 5to 7-membered heterocyclics. For example, proline analogs can be made inwhich the ring size of the proline residue is changed from a 5-memberedring to a 4-, 6-, or 7-membered ring. Cyclic groups can be saturated orunsaturated, and if unsaturated, can be aromatic or non-aromatic.Heterocyclic groups can contain one or more nitrogen, oxygen, and/orsulphur heteroatoms. Examples of such groups include furazanyl, furyl,imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g.,1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,thiomorpholino), and triazolyl groups. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl. Peptides, as well as peptide analogs and mimetics,can also be covalently bound to one or more of a variety ofnonproteinaceous polymers, for example, polyethylene glycol,polypropylene glycol, or polyoxyalkenes, as described in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337.

Other peptide analogs and mimetics within the scope of the disclosureinclude glycosylation variants, and covalent or aggregate conjugateswith other chemical moieties. Covalent derivatives can be prepared bylinkage of functionalities to groups which are found in amino acid sidechains or at the N- or C-termini, by means which are well known in theart. These derivatives can include, without limitation, aliphatic estersor amides of the carboxyl terminus, or of residues containing carboxylside chains, O-acyl derivatives of hydroxyl group-containing residues,and N-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues (e.g., lysine or arginine). Acyl groups are selectedfrom the group of alkyl-moieties including C3 to C18 normal alkyl,thereby forming alkanoyl aroyl species. Also embraced are versions of anative primary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, for example,phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties,including ribosyl groups or cross-linking reagents.

In another embodiment, a detectable moiety can be linked to themulti-domain peptides or peptide analogs disclosed herein, creating apeptide/peptide analog-detectable moiety conjugate. Detectable moietiessuitable for such use include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. The detectable moieties contemplated for thepresent disclosure can include, but are not limited to, animmunofluorescent moiety (e.g., fluorescein, rhodamine, Texas red, andthe like), a radioactive moiety (e.g., ³H, ³²P, ¹²⁵I ³⁵S), an enzymemoiety (e.g., horseradish peroxidase, alkaline phosphatase), acolorimetric moiety (e.g., colloidal gold, biotin, colored glass orplastic, and the like). The detectable moiety can be liked to themulti-domain peptide or peptide analog at either the N- and/orC-terminus Optionally, a linker can be included between the multi-domainpeptide or peptide analog and the detectable moiety.

Means of detecting such moieties are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

In another embodiment, an additional functional domain or peptide can belinked to the multi-domain peptides or peptide analogs disclosed herein,creating a peptide/peptide analog-additional functional domain/peptideconjugate. The additional functional domain or peptide can be linked tothe multi-domain peptide or peptide analog at either the N- and/orC-terminus. Optionally, a linker can be included between themulti-domain peptide or peptide analog and the additional functionaldomain or peptide. The additional functional domain or peptide canenhance the ability of the multi-domain peptide or peptide analog toefflux lipids from cells in a non-cytotoxic manner, and/or enhance itstherapeutic efficacy. Exemplary additional functional domains/peptidesinclude those shown in Table 5.

TABLE 5 Exemplary additional functional domains. Functional Domainor Peptide Sequence Cell recognition sequences Heparin binding  RKNR;site (SEQ ID NO: 46) KKWVR (SEQ ID NO: 47) Integrin binding RGD (and variants) site (SEQ ID NO: 48) P-selectin site DVEWVDVSY(SEQ ID NO: 49) Internalization sequences TAT HIV sequence RKKRRQRRRPPQ;(SEQ ID NO: 50) RRRQRRKKR (SEQ ID NO: 51) Panning sequence RRPXR(SEQ ID NO: 52) Penatratin sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 53)Neutral cholesterol  esterase activation SAA C-terminusGHEDTMADQEANRHGRSGGDPNYYRPPGGY sequence (SEQ ID NO: 54)Inhibition of ACAT SAA N-terminus GFFSFIGEAFQGAGDMWRAY sequence(SEQ ID NO: 55) Increase liver affinity LDL receptor KAEYKKNKHRH;sequence (SEQ ID NO: 56) YTRLTRKRGLK (SEQ ID NO: 57) Anti-oxidantactivity Modified 18A DWLKAFYCKVAEKLKEAF; sequence (SEQ ID NO: 58)DWLKAFYDKVAEKLKCAF (SEQ ID NO: 59) ApoA-I Milano YSDGLRQCLAARLDALKDRsequence (SEQ ID NO: 60) Heavy metal chelation 6x-His sequence HHHHHH(SEQ ID NO: 61) Lactoferrin FQWQRNIRKVR sequence (SEQ ID NO: 62)Lipoprotein Lipase  activation sequence  apoC-II  xxYxxIxxDQ;(residue 61-70) (SEQ ID NO: 69) wherein x is any amino acidthat will allow the domain to  activate lipoprotein lipase

Cell recognition sequences can increase the ability of the multi-domainpeptides or peptide analogs containing these sequences to bind to cells,the prerequisite first step in ABCA1-mediated cholesterol efflux(Remaley et al., Biochem. Biophys. Res. Commun. 280:818-823, 2001). Cellinternalization sequences, can increase the cellular uptake of themulti-domain peptides or peptide analogs into intracellularcompartments, where the initial lipidation of the peptides has beenproposed to occur (Neufeld et al., J. Biol. Chem. 279:15571-15578,2004), thus facilitating lipid efflux. Sequences that activate neutralcholesterol hydrolase (Kisilevsky et al., J. Lipid Res. 44:2257-2269,2003) can increase the amount of intracellular free cholesterol, theform of cholesterol that effluxes from cells. Similarly, the inhibitionof ACAT blocks the esterification of cholesterol to cholesteryl ester,thus increasing the pool of free cholesterol for efflux by themulti-domain peptides or peptide analogs (Kisilevsky et al., J. LipidRes. 44:2257-2269, 2003). Sequences that target the multi-domainpeptides or peptide analogs to the liver can facilitate the last step ofreverse cholesterol transport, the hepatic uptake and excretion ofcholesterol into the bile (Collet et al., J. Lipid Res. 40:1185-1193,1999). Part of the beneficial effect of apoA-I and synthetic peptidemimics is believed to be due to their anti-inflammatory and anti-oxidantproperties (Van Lenten et al., J. Clin. Invest. 96:2758-2767, 1995).Sequences containing domains that sequester oxidized lipids (Datta etal., J. Biol. Chem. 279:26509-26517, 2004), that act as antioxidants(Bielicki et al., Biochem. 41:2089-2096, 2002), or that chelate heavymetals (Wakabayashi et al., Biosci. Biotechnol. Biochem. 63:955-957,1999), which promote lipid oxidation, can compliment the lipid effluxproperties of the multi-domain peptides or peptide analogs by alsopreventing lipid oxidation. Lipoprotein lipase activation sequences canprevent, reduce or inhibit hypertriglyceridemia associated withadministration of any of the disclosed multi-domain peptides or peptideanalogs. In an example, lipoprotein lipase activation sequences resultin triglyceride levels of no greater than 200 mg/dl.

LPL activation can be determined by methods known to those of skill inthe art. In a particular example, an assay mixture of 200 μl containing2 mg of triacylglycerols from a lipid emulsion can be used. The mediumcan contain 0.1 M NaCl, 0.1 M Tris-Cl, 20 μg of heparin, and 12 mg ofbovine serum albumin (pH 8.5). To study the activation, peptides can bedissolved in 5 M urea, 10 mM Tris-Cl (pH 8.2). Known activators of LPL,such as apoC-II, can be used as a positive control. The proteinconcentration can be determined by a bicinchoninic acid protein assay.Five μl of the stock solution of apoCIl, or the same volume of dilutionsin 5 M urea, 10 mM Tris (pH 8.2), can be added to the incubations. Thereactions can be started by addition of LPL and stopped after incubationfor 15 min at 25° C. by addition of organic solvents for extraction ofthe labeled free fatty acids. The lipase activity can be expressed inunits/mg of LPL, where 1 unit corresponds to release of 1 μmol of fattyacid per min. An at least 10% increase in LPL activity, which is morethan 3× the coefficient of variation of most LPL assay, in the presenceof a test peptide as compared to LPL activity in the absence of the testpeptide, identifies the peptide as an LPL activating peptide.

In particular embodiments, the ability of the test peptides to activateLPL in vivo, can be determined by administering one or more of thepeptides. Triglycerides levels in a sample taken from a subjectreceiving the peptide alone can then be compared to those levels presentin a sample taken prior to the subject receiving the peptide or comparedto a reference sample containing a known amount of triglycerides.Triglyceride levels of less than 150 mg/dl following peptideadministration indicate that the peptide is an LPL activator or apeptide that causes a statistically significant decrease in the baselinetriglyceride level when tested in vivo. The linkers contemplated by thepresent disclosure can be any bifunctional molecule capable ofcovalently linking two peptides to one another. Thus, suitable linkersare bifunctional molecules in which the functional groups are capable ofbeing covalently attached to the N- and/or C-terminus of a peptide.Functional groups suitable for attachment to the N- or C-terminus ofpeptides are well known in the art, as are suitable chemistries foreffecting such covalent bond formation.

The linker may be flexible, rigid or semi-rigid. Suitable linkersinclude, for example, amino acid residues such as Pro or Gly or peptidesegments containing from about 2 to about 5, 10, 15, 20, or even moreamino acids, bifunctional organic compounds such as H₂N(CH₂)_(n)COOHwhere n is an integer from 1 to 12, and the like. Examples of suchlinkers, as well as methods of making such linkers and peptidesincorporating such linkers, are well-known in the art (see, e.g., Huniget al., Chem. Ber. 100:3039-3044, 1974 and Basak et al., Bioconjug.Chem. 5:301-305, 1994).

Conjugation methods applicable to the present disclosure include, by wayof non-limiting example, reductive amination, diazo coupling, thioetherbond, disulfide bond, amidation and thiocarbamoyl chemistries. In oneembodiment, the amphipathic α-helical domains are “activated” prior toconjugation. Activation provides the necessary chemical groups for theconjugation reaction to occur. In one specific, non-limiting example,the activation step includes derivatization with adipic aciddihydrazide. In another specific, non-limiting example, the activationstep includes derivatization with the N-hydroxysuccinimide ester of3-(2-pyridyl dithio)-propionic acid. In yet another specific,non-limiting example, the activation step includes derivatization withsuccinimidyl 3-(bromoacetamido) propionate. Further, non-limitingexamples of derivatizing agents include succinimidylformylbenzoate andsuccinimidyllevulinate.

V. Synthesis and Purification of the Peptides

The single and multi-domain peptides or peptide analogs of thedisclosure can be prepared using virtually any technique known to one ofordinary skill in the art for the preparation of peptides. For example,the peptides can be prepared using step-wise solution or solid phasepeptide syntheses, or recombinant DNA techniques, or the equivalentsthereof.

A. Chemical Synthesis

Peptides of the disclosure having either the D- or L-configuration canbe readily synthesized by automated solid phase procedures well known inthe art. Suitable syntheses can be performed by utilizing “T-boc” or“F-moc” procedures. Techniques and procedures for solid phase synthesisare described in Solid Phase Peptide Synthesis: A Practical Approach, byE. Atherton and R. C. Sheppard, published by IRL, Oxford UniversityPress, 1989. Alternatively, the multi-domain peptides may be prepared byway of segment condensation, as described, for example, in Liu et al.,Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc.117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res.45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu andTam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl.Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. PeptideProtein Res. 31:322-334, 1988). This is particularly the case withglycine containing peptides. Other methods useful for synthesizing themulti-domain peptides of the disclosure are described in Nakagawa etal., J. Am. Chem. Soc. 107:7087-7092, 1985.

Additional exemplary techniques known to those of ordinary skill in theart of peptide and peptide analog synthesis are taught by Bodanszky, M.and Bodanszky, A., The Practice of Peptide Synthesis, Springer Verlag,New York, 1994; and by Jones, J., Amino Acid and Peptide Synthesis, 2nded., Oxford University Press, 2002. The Bodanszky and Jones referencesdetail the parameters and techniques for activating and coupling aminoacids and amino acid derivatives. Moreover, the references teach how toselect, use and remove various useful functional and protecting groups.

Peptides of the disclosure having either the D- or L-configuration canalso be readily purchased from commercial suppliers of syntheticpeptides. Such suppliers include, for example, Advanced ChemTech(Louisville, Ky.), Applied Biosystems (Foster City, Calif.), Anaspec(San Jose, Calif.), and Cell Essentials (Boston, Mass.).

B. Recombinant Synthesis

If the peptide is composed entirely of gene-encoded amino acids, or aportion of it is so composed, the multi-domain peptide or the relevantportion can also be synthesized using conventional recombinant geneticengineering techniques. For recombinant production, a polynucleotidesequence encoding the multi-domain peptide is inserted into anappropriate expression vehicle, that is, a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence, or in the case of an RNA viral vector, the necessaryelements for replication and translation. The expression vehicle is thentransfected into a suitable target cell which will express themulti-domain peptide. Depending on the expression system used, theexpressed peptide is then isolated by procedures well-established in theart. Methods for recombinant protein and peptide production are wellknown in the art (see, e.g., Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, Ch. 17 and Ausubel et al. ShortProtocols in Molecular Biology, 4^(th) ed., John Wiley & Sons, Inc.,1999).

To increase efficiency of production, the polynucleotide can be designedto encode multiple units of the multi-domain peptide separated byenzymatic cleavage sites. The resulting polypeptide can be cleaved(e.g., by treatment with the appropriate enzyme) in order to recover thepeptide units. This can increase the yield of peptides driven by asingle promoter. In one embodiment, a polycistronic polynucleotide canbe designed so that a single mRNA is transcribed which encodes multiplepeptides, each coding region operatively linked to a cap-independenttranslation control sequence, for example, an internal ribosome entrysite (IRES). When used in appropriate viral expression systems, thetranslation of each peptide encoded by the mRNA is directed internallyin the transcript, for example, by the IRES. Thus, the polycistronicconstruct directs the transcription of a single, large polycistronicmRNA which, in turn, directs the translation of multiple, individualpeptides. This approach eliminates the production and enzymaticprocessing of polyproteins and can significantly increase yield ofpeptide driven by a single promoter.

A variety of host-expression vector systems may be utilized to expressthe peptides described herein. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus(TMV)) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing an appropriate coding sequence; or animal cellsystems.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, can be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like can be used. When cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter can beused. When cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters, the promoter for thesmall subunit of RUBISCO, the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV, thecoat protein promoter of TMV) can be used. When cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter, the vaccinia virus 7.5 K promoter) can beused.

C. Purification

The peptides or peptide analogs of the disclosure can be purified bymany techniques well known in the art, such as reverse phasechromatography, high performance liquid chromatography, ion exchangechromatography, size exclusion chromatography, affinity chromatography,gel electrophoresis, and the like. The actual conditions used to purifya particular multi-domain peptide or peptide analog will depend, inpart, on synthesis strategy and on factors such as net charge,hydrophobicity, hydrophilicity, and the like, and will be apparent tothose of ordinary skill in the art.

For affinity chromatography purification, any antibody whichspecifically binds the multi-domain peptide or peptide analog may beused. For the production of antibodies, various host animals, includingbut not limited to, rabbits, mice, rats, and the like, may be immunizedby injection with a multi-domain peptide or peptide analog. Themulti-domain peptide or peptide analog can be attached to a suitablecarrier (e.g., BSA) by means of a side chain functional group or linkerattached to a side chain functional group. Various adjuvants may be usedto increase the immunological response, depending on the host species,including but not limited to, Freund's (complete and incomplete),mineral gels (e.g., aluminum hydroxide), surface active substances(e.g., lysolecithin, pluronic polyols, polyanions, and oil emulsions),keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacteriumparvum.

Booster injections can be given at regular intervals, and antiserumharvested when the antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, e.g.,Ouchterlony et al., Handbook of Experimental Immunology, Wier, D. (ed.),Chapter 19, Blackwell, 1973. A plateau concentration of antibody isusually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM).Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher (Manualof Clinical Immunology, Ch. 42, 1980).

Monoclonal antibodies to a peptide or peptide analog may be preparedusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture, for example the classicmethod of Kohler & Milstein (Nature 256:495-97, 1975), or a derivativemethod thereof. Briefly, a mouse is repetitively inoculated with a fewmicrograms of the selected protein immunogen (e.g., a multi-domainpeptide or peptide analog) over a period of a few weeks. The mouse isthen sacrificed, and the antibody-producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and the excess unfused cells destroyed bygrowth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as enzyme-linked immunosorbent assay(ELISA), as originally described by Engvall (Meth. Enzymol., 70:419-39,1980), or a derivative method thereof. Selected positive clones can beexpanded and their monoclonal antibody product harvested for use.Detailed procedures for monoclonal antibody production are described inHarlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York,1999. Polyclonal antiserum containing antibodies can be prepared byimmunizing suitable animals with a polypeptide comprising at least onemulti-domain peptide or peptide analog, which can be unmodified ormodified, to enhance immunogenicity.

Antibody fragments may be used in place of whole antibodies and may bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as “antibody fragments,” are well known and include thosedescribed in Better & Horowitz, Methods Enzymol. 178:476-96, 1989;Glockshuber et al., Biochemistry 29:1362-67, 1990; and U.S. Pat. Nos.5,648,237 (Expression of Functional Antibody Fragments); 4,946,778(Single Polypeptide Chain Binding Molecules); and 5,455,030(Immunotherapy Using Single Chain Polypeptide Binding Molecules), andreferences cited therein. Conditions whereby a polypeptide/binding agentcomplex can form, as well as assays for the detection of the formationof a polypeptide/binding agent complex and quantitation of bindingaffinities of the binding agent and polypeptide, are standard in theart. Such assays can include, but are not limited to, Western blotting,immunoprecipitation, immunofluorescence, immunocytochemistry,immunohistochemistry, fluorescence activated cell sorting (FACS),fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA,ELISPOT (Coligan et al., Current Protocols in Immunology, Wiley, NY,1995), agglutination assays, flocculation assays, cell panning, etc., asare well known to one of skill in the art.

VI. Pharmaceutical Compositions and Uses Thereof

The peptides or peptide analogs of the disclosure (and mixtures thereof)can be used to treat any disorder in animals, especially mammals (e.g.,humans), for which promoting lipid efflux is beneficial, includingpromoting lipid efflux without inducing hypertriglyceridemia or withreduced hypertriglyceridemia. Such conditions include, but are notlimited to, hyperlipidemia (e.g., hypercholesterolemia), cardiovasculardisease (e.g., atherosclerosis), restenosis (e.g., atheroscleroticplaques), peripheral vascular disease, acute coronary syndrome,reperfusion myocardial injury, and the like. The peptides or peptideanalogs of the disclosure can also be used during the treatment ofthrombotic stroke and during thrombolytic treatment of occluded coronaryartery disease. In some examples, peptides or peptide analogs includinglipid efflux activity and lipoprotein lipase activating activity, suchas a peptide with an amino acid sequence provided by any one of SEQ IDNOs: 70-75, can be used to treat any of the aforementioned disorders.

The peptides or peptide analogs can be used alone or in combinationtherapy with other lipid lowering compositions or drugs used to treatthe foregoing conditions, or with agents (such as peptides) thatactivate LPL activity. Such combination therapies include, but are notlimited to simultaneous or sequential administration of the drugsinvolved. For example, in the treatment of hypercholesterolemia oratherosclerosis, the peptide or peptide analog formulations can beadministered with any one or more of the cholesterol lowering therapiescurrently in use, for example, bile-acid resins, niacin and statins.

In other embodiments, the multi-domain peptide or peptide analogformulations can be administered with a lipoprotein lipase activatingagent, such as an apoC-II protein, variant or fragment thereof, toprevent, reduce or inhibit hypertriglyceridemia associated with theadministration of any of the disclosed peptides or peptide analogs.

In another embodiment, the multi-domain peptides or peptide analogs canbe used in conjunction with statins or fibrates to treat hyperlipidemia,hypercholesterolemia and/or cardiovascular disease, such asatherosclerosis. In yet another embodiment, the multi-domain peptides orpeptide analogs of the disclosure can be used in combination with ananti-microbials agent and/or an anti-inflammatory agent. In a furtherembodiment, the multi-domain peptides can also be expressed in vivo, byusing any of the available gene therapy approaches.

A. Administration of Peptides or Peptide Analogs

In some embodiments, multi-domain peptides or peptide analogs can beisolated from various sources and administered directly to the subject.For example, a multi-domain peptide or peptide analog can be expressedin vitro, such as in an E. coli expression system, as is well known inthe art, and isolated in amounts useful for therapeutic compositions.

In exemplary applications, therapeutic compositions are administered toa subject suffering from a dyslipidemic or vascular disorder, such ashyperlipidemia, hyperlipoproteinemia, hypercholesterolemia,hypertriglyceridemia, HDL deficiency, apoA-I deficiency, coronary arterydisease, atherosclerosis, thrombotic stroke, peripheral vasculardisease, restenosis, acute coronary syndrome, or reperfusion myocardialinjury, in an amount sufficient to inhibit or treat the dyslipidemic orvascular disorder. Amounts effective for this use will depend upon theseverity of the disorder and the general state of the subject's health.A therapeutically effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

A multi-domain peptide or peptide analog can be administered by anymeans known to one of skill in the art (see, e.g., Banga, “ParenteralControlled Delivery of Therapeutic Peptides and Proteins,” inTherapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,Lancaster, Pa., 1995), such as by intramuscular, subcutaneous, orintravenous injection, but even oral, nasal, or anal administration iscontemplated. In one embodiment, administration is by subcutaneous orintramuscular injection. To extend the time during which themulti-domain peptide or peptide analog is available to inhibit or treata dyslipidemic or vascular disorder, the multi-domain peptide or peptideanalog can be provided as an implant, an oily injection, or as aparticulate system. The particulate system can be a microparticle, amicrocapsule, a microsphere, a nanocapsule, or similar particle (Banga,“Parenteral Controlled Delivery of Therapeutic Peptides and Proteins,”in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,Lancaster, Pa., 1995).

In one specific, non-limiting example, a multi domain peptide isadministered that includes one or more of the amino acid sequences shownin SEQ ID NOs: 3-45 and 70-75. In another specific, non-limitingexample, a multi domain peptide is administered that includes one ormore of the amino acid sequences shown in SEQ ID NOs: 3-45, whichpromotes lipid efflux, in combination with a lipoprotein lipaseactivating agent, such as an apoC-II protein, fragment or variantthereof (e.g., a peptide with an amino acid sequence set forth in anyone of SEQ ID NOs: 63-69); such LPL activating agent need not becovalently linked, or even administered simultaneously with, the peptidethat promotes lipid efflux.

B. Administration of Nucleic Acid Molecules

In some embodiments where the therapeutic agent is composed entirely ofgene-encoded amino acids, or a portion of it is so composed,administration of the multi-domain peptide or mixture of peptides, orthe relevant portion, can be achieved by an appropriate nucleic acidexpression vector (or combination of vectors) which is administered sothat it becomes intracellular, for example, by use of a retroviralvector (see U.S. Pat. No. 4,980,286), or by direct injection, or by useof microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.,88:1864-1868, 1991). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression,for example, by homologous or non-homologous recombination.

Use of a DNA expression vector (e.g., the vector pCDNA) is an example ofa method of introducing the foreign cDNA into a cell under the controlof a strong viral promoter (e.g., cytomegalovirus) to drive theexpression. However, other vectors can be used. Other retroviral vectors(such as pRETRO-ON, BD Biosciences, Palo Alto, Calif.) also use thispromoter but have the advantages of entering cells without anytransfection aid, integrating into the genome of target cells only whenthe target cell is dividing. It is also possible to turn on theexpression of a therapeutic nucleic acid by administering tetracyclinewhen these plasmids are used. Hence these plasmids can be allowed totransfect the cells, then administer a course of tetracycline to achieveregulated expression.

Other plasmid vectors, such as pMAM-neo (BD Biosciences, Palo Alto,Calif.) or pMSG (Invitrogen, Carlsbad, Calif.) use the MMTV-LTR promoter(which can be regulated with steroids) or the SV10 late promoter (pSVL,Invitrogen, Carlsbad, Calif.) or metallothionein-responsive promoter(pBPV, Invitrogen, Carlsbad, Calif.) and other viral vectors, includingretroviruses. Examples of other viral vectors include adenovirus, AAV(adeno-associated virus), recombinant HSV, poxviruses (vaccinia) andrecombinant lentivirus (such as HIV). All these vectors achieve thebasic goal of delivering into the target cell the cDNA sequence andcontrol elements needed for transcription.

Retroviruses have been considered a preferred vector for gene therapy,with a high efficiency of infection and stable integration andexpression (Orkin et al., Prog. Med. Genet. 7:130-142, 1988). A nucleicacid encoding the multi-domain peptide can be cloned into a retroviralvector and driven from either its endogenous promoter (where applicable)or from the retroviral LTR (long terminal repeat). Other viraltransfection systems may also be utilized for this type of approach,including adenovirus, AAV (McLaughlin et al., J. Virol. 62:1963-1973,1988), vaccinia virus (Moss et al., Annu. Rev. Immunol. 5:305-324,1987), Bovine Papilloma virus (Rasmussen et al., Methods Enzymol.139:642-654, 1987) or members of the herpesvirus group such asEpstein-Barr virus (Margolskee et al., Mol. Cell. Biol. 8:2837-2847,1988).

In addition to delivery of a nucleic acid encoding the multi-domainpeptide to cells using viral vectors, it is possible to usenon-infectious methods of delivery. For instance, lipidic andliposome-mediated gene delivery has recently been used successfully fortransfection with various genes (for reviews, see Templeton and Lasic,Mol. Biotechnol., 11:175-180, 1999; Lee and Huang, Crit. Rev. Ther. DrugCarrier Syst., 14:173-206, 1997; and Cooper, Semin. Oncol., 23:172-187,1996). For instance, cationic liposomes have been analyzed for theirability to transfect monocytic leukemia cells, and shown to be a viablealternative to using viral vectors (De Lima et al., Mol. Membr. Biol.,16:103-109, 1999). Such cationic liposomes can also be targeted tospecific cells through the inclusion of, for instance, monoclonalantibodies or other appropriate targeting ligands (Kao et al., CancerGene Ther., 3:250-256, 1996).

C. Representative Methods of Administration, Formulations and Dosage

The provided multi-domain peptides or peptide analogs, constructs, orvectors encoding such peptides, can be combined with a pharmaceuticallyacceptable carrier (e.g., a phospholipid or other type of lipid) orvehicle for administration to human or animal subjects. In someembodiments, more than one multi-domain peptide or peptide analog can becombined to form a single preparation. The multi-domain peptides orpeptide analogs can be conveniently presented in unit dosage form andprepared using conventional pharmaceutical techniques. Such techniquesinclude the step of bringing into association the active ingredient andthe pharmaceutical carrier(s) or excipient(s). In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing adose or unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, formulations encompassed herein mayinclude other agents commonly used by one of ordinary skill in the art.

The pharmaceutical compositions provided herein, including those for usein treating dyslipidemic and vascular disorders, may be administeredthrough different routes, such as oral, including buccal and sublingual,rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous,intradermal, and topical. They may be administered in different forms,including but not limited to solutions, emulsions and suspensions,microspheres, particles, microparticles, nanoparticles, and liposomes.In one embodiment, multi-domain peptides or peptide analogs withsuitable features of ABCA1-specificity and low cytotoxicity can beprecomplexed with phospholipids or other lipids into either discoidal orspherical shape particles prior to administration to subjects.

In another embodiment, it may be desirable to administer thepharmaceutical compositions locally to the area in need of treatment.This may be achieved by, for example, and not by way of limitation,local or regional infusion or perfusion during surgery, topicalapplication (e.g., wound dressing), injection, catheter, suppository, orimplant (e.g., implants formed from porous, non-porous, or gelatinousmaterials, including membranes, such as sialastic membranes or fibers),and the like.

In a specific embodiment, one or more of the disclosed peptides capableof promoting lipid efflux and activating lipoprotein lipase may beassociated either by coating or impregnating an implant such as stent totreat a dyslipidemic or vascular disorder. These peptides are preparedand purified as described herein. In an example, the implant can bepartially or completely coated with the peptide. For instance, theluminal surface of the implant may be coated with the peptide. Suchconfiguration is believed to reduce atherosclerotic plaques in arteriesoften associated with atherosclerosis while minimizing the amount ofcoating material and time required to prepare the implant. The peptidemay be attached to the implant by any chemical or mechanical bond orforce, including linking agents. Alternatively, the coating may bedirectly linked (tethered) to the first surface, such as through silanegroups. In other examples, the implant may be impregnated with at leastone peptide by methods known to those of skill in the art so thatmultiple surfaces (such as the outer and inner surfaces) of the implantinclude the peptide.

In an additional embodiment, the implant may be coated or impregnatedwith materials in addition to the disclosed peptides to further enhancetheir bio-utility. Examples of suitable coatings are medicated coatings,drug-eluting coatings, hydrophilic coatings, smoothing coatings.

In one embodiment, administration can be by direct injection at the site(or former site) of a tissue that is to be treated, such as the heart orthe peripheral vasculature. In another embodiment, the pharmaceuticalcompositions are delivered in a vesicle, in particular liposomes (see,e.g., Langer, Science 249:1527-1533, 1990; Treat et al., in Liposomes inthe Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, N.Y., pp. 353-365, 1989).

In yet another embodiment, the pharmaceutical compositions can bedelivered in a controlled release system. In one embodiment, a pump canbe used (see, e.g., Langer Science 249:1527-1533, 1990; Sefton Crit.Rev. Biomed. Eng. 14:201-240, 1987; Buchwald et al., Surgery 88:507-516,1980; Saudek et al., N. Engl. J. Med. 321:574-579, 1989). In anotherembodiment, polymeric materials can be used (see, e.g., Ranger et al.,Macromol. Sci. Rev. Macromol. Chem. 23:61-64, 1983; Levy et al., Science228:190-192, 1985; During et al., Ann. Neurol. 25:351-356, 1989; andHoward et al., J. Neurosurg. 71:105-112, 1989). Other controlled releasesystems, such as those discussed in the review by Langer (Science249:1527-1533, 1990), can also be used.

The amount of the pharmaceutical compositions that will be effectivedepends on the nature of the disorder or condition to be treated, aswell as the stage of the disorder or condition. Effective amounts can bedetermined by standard clinical techniques. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and should be decided according to the judgment of thehealth care practitioner and each subject's circumstances. An example ofsuch a dosage range is 0.1 to 200 mg/kg body weight in single or divideddoses. Another example of a dosage range is 1.0 to 100 mg/kg body weightin single or divided doses.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, and severity of the condition of the subjectundergoing therapy.

The pharmaceutical compositions of the present disclosure can beadministered at about the same dose throughout a treatment period, in anescalating dose regimen, or in a loading-dose regime (e.g., in which theloading dose is about two to five times the maintenance dose). In someembodiments, the dose is varied during the course of a treatment basedon the condition of the subject being treated, the severity of thedisease or condition, the apparent response to the therapy, and/or otherfactors as judged by one of ordinary skill in the art. The volume ofadministration will vary depending on the route of administration. Byway of example, intramuscular injections may range from about 0.1 ml toabout 1.0 ml. Those of ordinary skill in the art will know appropriatevolumes for different routes of administration.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1 Lipid Efflux from Cells Mediated by SyntheticPeptides

This example demonstrates the ability of synthetic peptides containingamphipathic helices to efflux lipid from ABCA1-expressing cells.

HeLa cells stably transfected with human ABCA1 cDNA (ABCA1 cells) andHeLa cells transfected with only a hygromycin-resistant control plasmid(control cells) were produced and grown in α-modified Eagle's medium(αMEM) plus 10% fetal calf serum, as described by Remaley et al.(Biochem. Biophys. Res. Commun. 280:818-823, 2001). Cholesterol andphospholipid efflux was performed for 18 hours on noncholesterol-loadedcells radiolabeledwith either cholesterol or choline (Remaley et al.,Arterioscler. Thromb. Vasc. Biol. 17:1813-1821, 1997). Percentage effluxwas calculated after subtracting the radioactive counts in the blankmedia (αMEM plus 1 mg/ml of BSA), and expressed as the percent of totalradioactive counts removed from the cells during the efflux period.

Cell fixation was performed by a 10 minute treatment with 3%paraformaldehyde in phosphate buffered saline (PBS), followed by threewashes with blank media. Lactate dehydrogenase (LDH) release from cellsinto the media was measured enzymatically (Roche Diagnostics,Indianapolis, Ind.) and expressed, after subtraction of LDH releasedinto blank media, as the percentage of total cell LDH. Total cell LDHwas determined after cell solubilization with 1% Triton X-100.

The 37pA peptide: DWLKAFYDKVAEKLKEAFPDWLKAFYDKVA EKLKEAF (SEQ ID NO: 1)was synthesized by a solid-phase procedure, using a Fmoc/DIC/HOBtprotocol on a Biosearch 9600 peptide synthesizer (Applied Biosystems,Foster City, Calif.). Both L-amino acid (L-37pA) and D-amino acid(D-37pA) enantiomers were synthesized. All peptides were purified togreater than 98% homogeneity by reverse-phase HPLC on an Aquapore RP-300column.

ABCA1 cells were used to assess the ability of apoA-I and syntheticpeptides to efflux lipid from cells (FIG. 1). As previously described(Hamon et al., Nat. Cell Biol. 2:399-406, 2000 and Remaley et al.,Biochem. Biophys. Res. Commun. 280:818-823, 2001), control cells do notefflux significant amounts of cholesterol and phospholipid to apoA-I,but do so after transfection with ABCA1 (FIGS. 1A, B). The L-37pApeptide, which was synthesized with all L-amino acids and only has twoamphipathic helices in contrast to the 10 present in apoA-I, effluxedapproximately 2- to 4-fold more cholesterol and phospholipid from ABCA1cells than from control cells (FIGS. 1C, D). Both the L-37pA peptide andapoA-I began to show saturation for lipid efflux at approximately thesame protein concentration of 10 μg/ml, but because the L-37pA peptideis significantly smaller in molecular weight than apoA-I, thiscorresponds to a molar concentration of 2 μM for L-37pA and 0.36 μM forapoA-I. The 37pA peptide synthesized with all D-amino acids, D-37pA, wasalso effective in promoting cholesterol and phospholipid efflux fromABCA1 cells (FIGS. 1E, F). D-37pA had a similar dose-response curve asL-37pA, suggesting that there is not a need for a stereoselectiveinteraction between the 37-pA peptide and the ABCA1 transporter forlipid efflux. Both L-37pA and D-37pA also consistently removed morecholesterol (5% at 40 μg/ml) and phospholipids (8% at 40 μg/ml) fromcontrol cells (FIG. 1C-F) than did apoA-I (FIGS. 1A, B).

Example 2 Lipid Efflux Time Course

This example demonstrates the cholesterol efflux time course fromABCA1-expressing cells to apoA-I and synthetic peptides containingamphipathic helices.

Cholesterol efflux from ABCA1 cells to apoA-I was first detectable after2 hours and continued to increase throughout the 30 hour efflux period(FIG. 2A). In contrast, there was no significant increase abovebackground in cholesterol efflux to apoA-I from control cells (FIG. 2B).Overall, the kinetics for cholesterol efflux to L-37pA from ABCA1 cellswas similar to that of apoA-I, except that cholesterol efflux was firstdetectable after 30 minutes (FIG. 2A). L-37pA peptide, unlike apoA-I,also promoted cholesterol efflux from control cells but at approximatelyhalf the rate (FIG. 2B). A small amount of cholesterol efflux to L-37pAfrom control cells was first detectable at 30 minutes, and then itslowly continued to increase throughout the efflux period, similar towhat was observed for L-37pA with ABCA1 cells.

Example 3 Importance of Amphipathic a Helices

This example demonstrates the importance of amphipathic a helices inpeptide-lipid affinity and in the ability of peptides to promote lipidefflux from cells.

The introduction of D-amino acids into a peptide that otherwise containsL-amino acids is known to interfere with the ability of a peptide toform an alpha helix (Chen et al., J. Pept. Res. 59:18-33, 2002). Inorder to test the importance of amphipathic alpha helices in peptidelipid affinity and in the ability of peptides to promote lipid effluxfrom cells, the following 2 peptides with the same sequence as 37pA weremade with a mixture of L- and D-amino acids: (1) L2D-37pA, all L-aminoacids except that D-amino acids were used for valine and tyrosine; and(2) L3D-37pA, all L-amino acids except that D-amino acids were used foralanine, lysine, and aspartic acid. The L2D-37pA and L3D-37pA peptideshad lower lipid affinity, as assessed by monitoring their ability to actas detergents in the solubilization of dimyristoyl phosphatidyl choline(DMPC) vesicles. The solubilization of multilamellar DMPC vesicles (2mg/ml) by the peptides (0.4 mg/ml) was performed in the presence of 8.5%NaBr, and the absorbance at 350 nm was measured after a 2 hourincubation at room temperature, as previously described (Jonas, Methodsof Enzymology 128:553-581, 1986). After the 2 hour incubation, theL-37pA and D-37pA peptides nearly completely solubilized the DMPCvesicles, whereas the L3D-37pA peptide caused only a minimal decrease inturbidity (FIG. 3). The L2D-37pA peptide and apoA-I caused anintermediate level of DMPC vesicle solubilization compared to the L-37pAand L3D-37pA peptides.

When the L2D-37pA peptide was tested for lipid efflux, the substitutionof D-amino acids for valine and tyrosine residues caused a greater than75% reduction in cholesterol and phospholipid efflux from ABCA1 cellscompared to the L-37pA peptide, which contains all L-amino acids(Compare FIG. 4 with FIGS. 1C, D). Even though lipid efflux was reducedfrom ABCA1 cells to the L2D-37pA peptide compared to apoA-1, the peptidestill retained some ability to efflux lipid from ABCA1 cells, but it wasunable, like apoA-I, to promote any lipid efflux from control cells(FIGS. 1A, B). In contrast, L3D-37pA, which caused only minimal DMPCvesicle solubilization (FIG. 3), was also unable to promote detectableamounts of lipid efflux from either ABCA1 cells or control cells (FIG.4). A peptide based on the gamma crystalline protein(RMRITERDDFRGQMSEITDDCPSLQDRFHLTEVHSLRVLEGS (SEQ ID NO: 2); Hay et al.,Biochem Biophys. Res. Commun. 146:332-338, 1987), which contains twonon-amphipathic alpha helices of approximately the same length as thehelices on 37pA, was tested and also found to be completely ineffectivein promoting cholesterol and phospholipid efflux from either cell line.These results are consistent with previous studies that demonstrated theimportance of the amphipathic alpha helix in promoting lipid efflux(see, e.g., Gillotte et al., J. Biol. Chem. 274:2021-2028, 1999 andGillotte et al., J. of Lipid Res. 39:1918-1928, 1998). However, therelative level of lipid efflux from the two cell lines (FIGS. 1 and 4)demonstrates that amphipathic helical peptides can promote lipid effluxin an ABCA1-dependent and an ABCA1-independent manner, although theexpression of ABCA1 is necessary for those apolipoproteins and peptides,such as apoA-I and L2D-37pA, with only moderate lipid affinity, asassessed by DMPC vesicle solubilization (FIG. 3).

Example 4 Evaluation of the ABCA1-Independent Lipid Efflux Pathway

This example demonstrates that amphipathic helical peptides with highlipid affinity can promote lipid efflux in an ABCA1-independent manner.

In order to confirm that the residual lipid efflux from the controlcells to L-37pA and D-37pA (see FIG. 1) was not due to a low level ofendogenous ABCA1, a Tangier disease fibroblast cell line with atruncated non-functional ABCA1 transporter (Remaley et al., Proc. Natl.Acad. Sci. USA 96:12685-12690, 1999) was evaluated for lipid efflux(FIG. 5). ApoA-I, L-37pA, and D-37pA all effluxed cholesterol fromnormal fibroblasts, but apoA-I did not efflux significant amounts ofcholesterol from Tangier disease fibroblasts (see also, Francis et al.,J. Clin. Invest. 96:78-87, 1995 and Remaley et al., Arterioscler.Thromb. Vasc. Biol. 17:1813-1821, 1997). In contrast, both L-37pA andD-37pA were still able to efflux cholesterol from Tangier diseasefibroblasts, albeit at a reduced level, thus confirming the ability ofthese peptides to efflux lipid from cells in the absence of ABCA1.

The ABCA1-independent pathway for lipid efflux was further evaluated byexamining the effect of cell fixation with paraformaldehyde oncholesterol efflux to apoA-I (A), L-37pA (L), and D-37pA (D) (FIG. 6).In addition, 0.02% of taurodeoxycholate (T) was also tested for lipidefflux after 1 hour, in order to determine if a sublytic concentrationof a simple detergent would also promote more lipid efflux from ABCA1cells than from control cells. As expected, based on the ATP requirementof the ABCA1 transporter (Dean et al., J. Lipid Res. 42:1007-1017, 2001;Mendez, J. Lipid Res. 38:1807-1821, 1997), fixation of ABCA1 cells withparaformaldehyde completely blocked the ability of apoA-I to effluxcholesterol (FIG. 6A). In contrast, cell fixation of ABCA1 cells onlypartially reduced cholesterol efflux to the L-37pA and D-37pA peptides;approximately 30% of the baseline cholesterol efflux was still retainedafter cell fixation. When cholesterol efflux was tested on non-fixedcontrol cells, the level of cholesterol efflux to L-37pA and D-37pA wassimilar to the level obtained with fixed ABCA1 cells (compare FIGS. 6Band 6A). Furthermore, unlike ABCA1 cells, fixation of control cells didnot further reduce cholesterol efflux to the two peptides (FIG. 6B).These results indicate that lipid efflux by the peptides from ABCA1cells occurs by both an ABCA1-dependent and an ABCA1-independentpathway, whereas lipid efflux from control cells only occurs by theABCA1-independent pathway, which is a passive, energy-independentprocess that does not require viable cells.

The addition of a relatively low concentration (0.02%) oftaurodeoxycholate to the cell culture efflux media for 1 hour did notalter the morphology of the cells, as assessed by light microscopy, butdid result in a small amount of cholesterol efflux from ABCA1 cells(FIG. 6A), which slightly increased after fixation. Approximately thesame amount of cholesterol efflux also occurred from control cells afterthe taurodeoxycholate treatment (FIG. 6B). Nearly identical results werealso obtained with several other detergents (TX-100, NP-40, CHAPS) whentested at sublytic concentrations. This indicates that ABCA1 promoteslipid efflux to amphipathic helical proteins but does not increase theoverall propensity of cells to efflux lipids to simple detergents.

The inability to completely block peptide mediated lipid efflux by cellfixation (FIG. 6) and the correlation between DMPC vesiclesolubilization by the peptides with lipid efflux (FIGS. 1 and 3),suggests that lipid efflux from control cells occurs as the result ofthe microsolubilization of the cell membrane lipids by thedetergent-like action of the amphipathic helices on the peptides. Themicrosolubilization of the plasma membrane of cells could, therefore, bepotentially cytotoxic, but no morphologic effect was observed on thecells after incubation with the peptides or apoA-I, during the effluxexperiments. Incubation of the cells with L-37pA and D-37pA at themaximum concentration and time used for the efflux studies (40 μg /mlfor 18 hours) did, however, consistently result in the release of asmall amount of LDH from both cell lines (control cells: L-37pA(6.1%±0.2), D-37pA (6.6%±0.1); ABCA1 cells: L-37pA (4.3%±0.04), D-37pA(5.7%±0.1)). In contrast, L2D-37pA, L3D-37pA, and apoA-1, which did notcause lipid efflux from control cells (FIGS. 2 and 3) and, therefore,appear to be incapable of effluxing lipid by the ABCA1-independentpathway, also did not cause any significant release of cell LDH abovebaseline (<0.5%) from either cell line.

Example 5 Competition of Peptides/apoA-I for Binding of radiolabeledL-37pA

This example demonstrates the lack of stereoselectivity in the bindingof the 37pA peptide to either ABCA1 cells or control cells.

The L-37pA peptide was labeled with ¹²⁵I using iodine monochloride.Confluent cells grown on 12-well plates were incubated for 3 hours at 4°C. with the indicated concentration of the unlabeled competitor peptidein αMEM media plus 10 mg/ml of BSA (FIG. 7). The cells were then washedthree times and incubated for 1 hour at 4° C. with 1 μg/ml of theradiolabeled L-37pA peptide dissolved in αMEM media plus 10 mg/ml ofBSA. Cells were washed three times, and cell bound counts weredetermined after solubilization with 0.1 N NaOH.

A two-step sequential competitive binding assay was performed in orderto prevent any potentially interfering interaction of the radiolabeledpeptide with the competitor proteins (Mendez et al., J. Clin. Invest.94:1698-1705, 1994). The cells were first incubated with the competitorproteins for 3 hours, washed, and then the cell binding of theradiolabeled L-37 peptide was measured. At 8 μM, the maximumconcentration tested, which is equivalent to the maximum peptide proteinconcentration of 40 μg /ml used in the lipid efflux studies (FIG. 1),the unlabelled L-37pA peptide blocked the binding of approximately 40%of the labeled L-37pA peptide (FIG. 7A). D-37pA was similarly effectivein competing for the binding of L-37pA, indicating a lack ofstereoselectivity in the binding of the peptides to ABCA1 cells.L3D-37pA, in contrast, was completely ineffective in competing for thebinding of L-37pA. L2D-37pA and apoA-I acted as intermediatecompetitors; they each reduced the binding of radiolabeled L-37pA toABCA1 cells by approximately 30% (FIG. 7A). Control cells also showedrelatively high specific binding of L-37pA (FIG. 7B), but in the absenceof a competitor, the control cells bound 23% less radiolabeled L-37pApeptide than ABCA1 cells (control cells 27±0.6 μmol/mg cell protein;ABCA1 cells 35±2.2 μmol/mg cell protein). Similar to ABCA1 cells,unlabelled L-37pA and D-37pA competed equally well for the binding ofradiolabeled L-37pA. In contrast, L2D-37pA and apoA-I were lesseffective in control cells than in ABCA1 cells for competing for thebinding of radiolabeled L-37pA. At the maximum concentration tested,both peptides blocked less than 5% of the radiolabeled L-37pA frombinding to control cells, similar to the result obtained with theinactive L3D-37pA peptide. Overall, these results indicate that there isa lack of stereoselectivity in the binding of the 37pA peptide to eitherABCA1 cells or control cells and that the cell binding of the peptidesis at least partly dependent upon their lipid affinity.

Example 6 Effect of Asymmetry in Lipid Affinity of Multi-DomainAmphipathic Peptides on Lipid Efflux and Cell Cytotoxicity

This example demonstrates that asymmetry in lipid affinity ofmulti-domain amphipathic peptides is an important structural determinantfor specificity of ABCA1-dependent cholesterol efflux by multi-domainpeptides.

The 37pA peptide was modified by making 5 Ala substitutions forhydrophobic residues (F18, L14, L3, V10, F6) in either the C-terminalhelix (5A) or both helices (10A). Reverse phase HPLC retention timesclosely correlated with their predicted lipid affinity, as calculated bythe hydrophobic moment of the modified peptides (FIG. 8). Fouradditional peptides with 1 (L14, 1A), 2 (L14, F18, 2A), 3 (L14, F18, F6,3A) and 4 (L14, F18, F6, V10, 4A) Ala substitutions in the C-terminalhelix were also synthesized. The 37pA had the longest retention time andwith each additional Ala substitution there was a decrease in lipidaffinity based on the retention time (FIG. 8).

The 37pA peptide and all of the modified peptides were then tested forcytotoxicity, using a red blood cell hemolysis assay (FIG. 9). Similarto results previously observed via monitoring LDH release, the 37pA wasfound to be cytotoxic. Approximately 25% of the red blood cells werelysed after 1 hour at the maximum dose tested (FIG. 9). Overall, themodified peptides containing the Ala substitutions were less cytotoxic,and the degree of cytotoxicity closely correlated with the number of Alasubstitutions. The 4A, and 5A peptides showed no appreciable hemolysisof the red blood cells, whereas the 1A, 2A and 3A peptides showed amoderate degree of hemolysis when compared to 37pA (FIG. 9). Based onthese results, the optimum hydrophobic moment score per residue for theamphipathic α-helix with relatively low lipid affinity, in terms ofreducing cytotoxicity, is less than about 0.34 (Eisenberg et al., PNAS81:140-144, 1984 and Eisenberg et al., J. Mol. Biol. 179:125-142, 1984).

The 37pA peptide and the modified peptides were also tested for theirspecificity for cholesterol efflux by the ABCA1 transporter (FIG. 10).The 37pA peptide promoted ABCA1-mediated cholesterol efflux, but it wasalso able to mediate cholesterol efflux from a control HeLa cell linethat does not express the ABCA1 transporter. When cholesterol efflux wasperformed with the modified peptides, they were observed to have twodifferent features than the 37pA peptide. First, there was a progressiverightward shift in the dose response curve with the Ala substitutionscompared to the 37pA peptide. A greater concentration of the modifiedpeptides was needed to get the maximum amount of cholesterol efflux. Inaddition, the percent of total cholesterol efflux attributable to theABCA1 transporter progressively increased by making the Alasubstitutions in the 37pA peptide. Without wishing to be bound bytheory, it is believed that this is due to the fact that the modifiedpeptides still retained their ability to remove cholesterol from theABCA1-transfected cells, but were less effective in removing cholesterolfrom the control cells via non-ABCA1 cholesterol efflux pathways. The 5Apeptide was completely specific for only causing cholesterol efflux bythe ABCA1 transporter. Based on these results, the optimum hydrophobicmoment score (Eisenberg scale; 100 degree-alpha helix) per residue forthe amphipathic helix with relatively low lipid affinity, in terms of ABCA1-specificity for cholesterol efflux, is between about 0.1 and about0.33.

Example 7 Identification of Non-Cytotoxic Peptides that PromoteAbca1-Dependent Lipid Efflux

This example illustrates a method for identifying non-cytotoxic peptidesthat promote ABCA1-dependent lipid efflux from cells.

Peptide Design Based on the principals and procedures described in thepresent application, an amino acid sequence can be designed for amulti-domain peptide that contains two or more amphipathic α-helices,one with relatively high lipid affinity and one with relatively lowlipid affinity.

Peptide production: Peptides to be tested can be produced syntheticallyor by recombinant DNA methods, as described in the present application,and purified by reverse phase HPLC or other suitable techniques wellknown to one of skill in the art.

Peptide Cytotoxicity Testing: Peptides can be tested for cytotoxicity byany number of methods well known to one of skill in the art, such as therelease of intracellular LDH (Example 4) or the release of hemoglobinfrom red blood cells (Example 6). Such studies are performed byincubating various concentrations of the peptides with a cell line, avesicle or red blood cells, as described herein.

Peptide ABCA1-specificity for Lipid Efflux: Peptides to be tested can beadded to serum-free cell culture media in the approximate concentrationrange of 1-20 micromolar and incubated with a control cell line thatdoes not express the ABCA1 transporter and the same cell line aftertransfection with human cDNA for the ABCA1 transporter, as describedherein. Alternatively, cells, such as macrophages, that either expressor do not express the ABCA1 transporter depending on their cholesterolcontent and/or exposure to agents that induce the ABCA1 transporter(e.g., cAMP and LXR agonists) can also be used. After a suitable periodof approximately 4 to 24 hours, the conditioned media can be removedfrom the cells and the amount of cholesterol and or phospholipideffluxed can be quantified, as described herein. ABCA1-specific lipidefflux is calculated by subtracting the total lipid efflux from theABCA1 expressing cell line from the results obtained from the cell linethat does not express the ABCA1 transporter.

Example 8 In Vivo Effects of 5A-Peptide in Mice

This example illustrates the in vivo effects of 5A-POPC(1-palmitoyl-2-oleoyl-sn-phosphatidylcholine) peptide in mice.

The 5A peptide (SEQ ID NO: 3) was complexed with POPC at a 1:7 molarratio using a co-lyophilization procedure by which the free peptide andPOPC were co-dissolved in glacial acetic acid and lypophilized. The drypowder was reconstituted with saline and heated between 37° C. to 50° C.to reconstitution, which results in a clear solution of particlescontaining both the 5A peptide and POPC.

Increasing concentrations (3.75 mg/kg; 7.5 mg/kg; 15 mg/kg; 30 mg/kg; 60mg/kg; 120 mg/kg; or 240 mg/kg) of 5A-POPC peptide were administeredintravenously to wild-type mice over a 24 hour period. Plasmacholesterol levels were then measured. As illustrated in FIG. 11, a doseresponse relationship between total cholesterol and the 5A peptide wasobserved. Based on fast protein liquid chromatography (FPLC) analysis,almost all of this increase in cholesterol was due to an increase in thecholesterol content of HDL, which is consistent with the ability of thepeptide to in vitro promote cholesterol efflux from cells.

As illustrated in FIG. 12, treatment of mice with the 5A peptide (SEQ IDNO: 3) reduced atherosclerosis. Treatment with the 5A peptide alsoappeared to be non-toxic based on measurement of various serumbiomarkers (FIG. 13). However, 5A peptide caused a transient increase intriglycerides (FIGS. 14 and 15). Similar effects on triglycerides havealso been measured for apoA-I and various apoA-I mimetic peptides.

Hypertriglyceridemia is a side effect of various drugs and can be due toliver toxicity, but it often occurs after chronic administration of thedrug and is persistent. In this case, the hypertriglyceridemia occurredalmost immediately after administration of the peptide and was transient(less than 8 hours) and there was no evidence of increased markers ofliver damage (FIG. 13). FPLC analysis indicated that the increase intriglycerides occurred on VLDL, which may be a consequence of theability of the 5A peptide to induce reverse cholesterol transport backto the liver and stimulate VLDL secretion.

Additional studies (see FIGS. 16 and 17) indicated that the increase intriglycerides was due to inhibition of lipoprotein lipase, the mainserum enzyme that hydrolyzes triglycerides on VLDL and chylomicrons.First, as illustrated in FIG. 15, the increase in triglycerides wassignificant in non-fasting mice (i.e., over a 10-fold increase wasobserved and the reactant level of serum triglycerides exceeded 1000mg/dL). In contrast, triglyceride levels in fasting mice were onlyincreased by 3-fold (FIG. 14). As illustrated in FIG. 16, increasingdoses of the 5A peptide led to no significant changes in triglyceridessynthesis in mice treated with Triton-WR 1339 (a detergent that inhibitslipoprotein lipase). These studies suggest that the hypertriglyceridemiaobserved after 5A injection resulted from decreased catabolism ofapoB-containing lipoproteins.

In the saline treated mice, there was a time dependent increase intriglycerides. When the mice were also treated with the 5A peptide therewas also an increase in triglycerides but compared to mice not treatedwith Triton-X (FIGS. 14 and 15), the increase over the saline controlwas not as large and was not statistically significant. This would beexpected if both the 5A peptide and Triton-X inhibit lipoprotein lipase.In FIG. 17, the direct effect of the 5A peptide was tested onlipoprotein lipase, using an in vitro assay. Using doses similar tothose which caused hypertriglyceridemia in mice, it was found that the5A peptide was a potent inhibitor of lipoprotein lipase revealing themechanism by which 5A peptide caused hypertriglyceridemia.

These studies demonstrate that while 5A-POPC peptide stimulatescholesterol efflux in mice in vivo, it also inhibits lipoprotein lipaseleading to transient hypertriglyceridemia. These studies also reveal anew use of the 5A peptide as a lipoprotein lipase inhibitor.

Example 9 In Vivo Effects of 18A-C-11 Peptide in Mice

This example illustrates the ability of 18A-C-11 peptide to reducetriglycerides and cholesterol plasma levels in normal and dyslipidemicmice.

18A-C-11 peptide (SEQ ID NO: 70) was complexed with POPC at a 1:7 molarratio using a co-lyophilization procedure by which the free peptide andPOPC were co-dissolved in glacial acetic acid and lypophilized The drypowder was reconstituted with saline and heated between 37° C. to 50° C.to reconstitution, which results in a clear solution of particlescontaining both the 18A-C-11 peptide and POPC.

Hypertriglyceridemia was induced in C57B1/6 mice by a 2 mg (60 mg/kg)bolus IV injection of free 5A peptide (A). As illustrated in FIG. 18,when 1 mg (30 mg/kg) of the 18A-C-II peptide was co-injected with free5A peptide (X), the increase in plasma triglycerides was significantlyreduced. In addition, a single IV dose of 1 mg of the 18A-C-II:POPCpeptide lowered the plasma triglyceride levels in normal C57B1 mice (□)compared to saline (⋄). Additionally, a single dose of 30 mg/kg (1 mg)of 18A-C-II:POPC peptide significantly reduced plasma triglyceridelevels in ApoE knockout mice (76%) when compared to baseline values (seeFIG. 19).

In order to investigate the effects of the 18A-C-II peptide in plasmacholesterol of C57B1/6 mice, mice were injected with an IV bolus of 1 mg(30 mg/kg) of 18A-C-II:POPC and plasma cholesterol levels were monitoredfor 4 hours. As illustrated in FIG. 20, a significant reduction inplasma cholesterol of 52% and 80% was observed after 1 hour and 4 hoursof the treatment.

ApoE knockout and LDLR knockout mice are characterized by high plasmalevels of VLDL/IDL and LDL respectively. To evaluate the potential toreduce the plasma cholesterol levels, mice were injected IV in boluswith a dose of 1 mg (30 mg/kg) of the lipidated 18A-C-II:POPC in ApoE KO(⋄) and 0.5 mg (15 mg/kg) in LDLR KO mice (□). As shown in FIG. 21, asignificant reduction (36%) in serum cholesterol in ApoE KO mice wasobserved, while the low dose of the peptide in LDLR KO mice showed areduction of 22% in plasma cholesterol after 2 hours of the treatment.

The changes observed in plasma cholesterol and triglycerides werefurther analyzed by fast protein liquid chromatography (FPLC). Asillustrated in FIG. 22, a dose of 30 mg/kg of 18A-C-II:POPC (1:7)peptide lead to reduction of LDL-c, HDL-c (left panel) as well asVLDL-tg, LDL-tg and HDL-tg (right panel) at 1 and 4 hours aftertreatment. (LDL-c=LDL cholesterol; tg=triglycerides). Each samplerepresents a pool of 3 mice.

The effect of 18A-C-II peptide on cholesterol efflux was investigated byloading BHK and BHK-ABCA1 cells with ³H-cholesterol for 24 hours at 37°C. and then adding 20 ug/ml of the free 18A-C-II peptide as cholesterolacceptor to efflux for 18 hours. Percentage efflux was calculated bysubtracting the radioactive counts in the blank from the radioactivecounts in the presence of an acceptor and then dividing the result bythe sum of the radioactive counts in the medium plus the cell fraction.As shown in FIG. 23, a significant difference was observed in cells thatexpress the ABCA1 transporter indicating that ABCA1 is, at least, onemechanism mediating the cholesterol efflux to the 18A-C-II peptide.

These studies show that the 18A-C-II peptide produces plasma changes intriglycerides similar to the native human protein. The 18A-C-II peptidewas able to reduce and to prevent the increase of triglycerides inplasma of normal and dyslipidemic mice. The 18A-C-II peptide was alsoable to reduce plasma cholesterol levels in normal and dyslipidemicmice. The reduction of cholesterol and triglycerides was seen in alllipoproteins. The efflux mediated by the 18A-C-II peptide is, at least,in part mediated by the ABCA-1 transporter. Altogether, the reducedtriglycerides and cholesterol plasma levels observed is compatible to anathero-protective and to an anti-metabolic syndrome profile.

The ability of the 18A-C-II peptide in reducing triglycerides isbelieved to be useful to treat conditions that lead tohypertriglyceridemia such as pancreatitis, metabolic syndrome, LPLdeficiency, and high triglycerides induced by apoA-I mimetic therapies.Moreover, the cholesterol lowering effect is believed to be a potentialnew alternative treatment to conditions such as familialhypercholesterolemia and other high cholesterol dyslipidemias.

Example 10 Identification of Peptides that Promote ABCA1-Dependent LipidEfflux and Activate Lipoprotein Lipase

This example illustrates a method for identifying peptides that promoteABCA1-dependent lipid efflux from cells and activates lipoprotein lipasewithin cells.

Peptide Design Based on the principals and procedures described in thepresent application, an amino acid sequence can be designed for apeptide that promotes lipid efflux and is capable of activatinglipoprotein lipase. In one example, the peptide contains one amphipathicα-helix, with relatively high lipid affinity, and an apoC-II domain(such as those provided in Table 3 above). The apoC-II domain can be inthe form of an α-helix resulting in a peptide with two α-helices.Alternatively, the apoC-II domain can be incorporated into one or moreof the amphipathic α-helical domains of the multi-domain peptidesdesigned and produced according to methods disclosed herein (such asthose provided in Example 7).

Peptide production: Peptides to be tested can be produced syntheticallyor by recombinant DNA methods, as described in the present application,and purified by reverse phase HPLC or other suitable techniques wellknown to one of skill in the art. In a particular example, the peptideprovided in Table 4 can be synthesized and purified.

Peptide Cytotoxicity Testing: Peptides can be tested for cytotoxicity byany number of methods well known to one of skill in the art, such as therelease of intracellular LDH (Example 4), the release of hemoglobin fromred blood cells (Example 6) or evaluating the in vivo effects of thepeptide on various serum biomarkers (Example 8) or triglyceride levels.Such studies are performed by incubating various concentrations of thepeptides with a cell line, a vesicle or red blood cells, oradministrating various concentrations of the peptide to the subject asdescribed herein.

Peptide ABCA1-specificity for Lipid Efflux: Peptides to be tested can beadded to serum-free cell culture media in the approximate concentrationrange of 1-20 micromolar (μM) and incubated with a control cell linethat does not express the ABCA1 transporter and the same cell line aftertransfection with human cDNA for the ABCA1 transporter, as describedherein. Alternatively, cells, such as macrophages, that either expressor do not express the ABCA1 transporter depending on their cholesterolcontent and/or exposure to agents that induce the ABCA1 transporter(e.g., cAMP and LXR agonists) can also be used. After a suitable periodof approximately 4 to 24 hours, the conditioned media can be removedfrom the cells and the amount of cholesterol and or phospholipideffluxed can be quantified, as described herein. ABCA1-specific lipidefflux is calculated by subtracting the total lipid efflux from theABCA1 expressing cell line from the results obtained from the cell linethat does not express the ABCA1 transporter. To determine the ability ofpeptides to stimulate lipid efflux in vivo, increasing concentrations(3.75 mg/kg; 7.5 mg/kg; 15 mg/kg; 30 mg/kg; 60 mg/kg; 120 mg/kg; or 240mg/kg) of the peptide can be administered intravenously to wild-typemice over a 24 hour period. Plasma cholesterol levels can then bemeasured.

Peptide activation of lipoprotein lipase (LPL): For measurement of LPLactivity an assay mixture of 200 μl containing 2 mg of triacylglycerolsfrom the lipid emulsion can be used. The medium can contain 0.1 M NaCl,0.1 M Tris-Cl, 20 μg of heparin, and 12 mg of bovine serum albumin (pH8.5). To study the activation, peptides can be dissolved in 5 M urea, 10mM Tris-Cl (pH 8.2). Known activators of LPL, such as apoC-II, can beused as a positive control. The protein concentration can be determinedby a bicinchoninic acid protein assay. Five μl of the stock solution ofapoC-II, or the same volume of dilutions in 5 M urea, 10 mM Tris (pH8.2), can be added to the incubations. The reactions can be started byaddition of LPL and stopped after incubation for 15 min at 25° C. byaddition of organic solvents for extraction of the labeled free fattyacids. The lipase activity is expressed in units/mg of LPL, where 1 unitcorresponds to release of 1 μmol of fatty acid per minute. An at least10% increase in LPL activity and or a statistically significant increase(3× the CV of the lipase assay) in LPL activity in the presence of atest peptide as compared to LPL activity in the absence of the testpeptide, identifies the peptide as an LPL activating peptide. Theability of the test peptides to activate LPL in vivo, can be determinedby administering one or more of the peptides. Triglycerides levels in asample taken from a subject receiving the peptide alone can then becompared to those levels present in a sample taken prior to the subjectreceiving the peptide or compared to a reference sample containing aknown amount of triglycerides. Triglyceride levels of less than 150mg/dl following peptide administration indicate that the peptide is anLPL activator.

Example 11 Associating a Peptide Capable of Promoting Lipid Efflux andActivating Lipoprotein Lipase with an Implant

According to the teachings herein, one or more peptides that promoteABCA1 specific lipid efflux and/or activate LPL, or a combinationthereof, can be placed in a suitable container, such as a tissuemicrocapsule implant, and placed within a subject to allow continuous,slow release of one or more of the disclosed peptides. Such peptides caneither be used in the free state or after complexation with lipid.

Example 12 Method of Treating or Inhibiting a Dyslipidemic or VascularDisorder in a Subject

According to the teachings herein, one or more of the disclosed peptidescapable of stimulating cholesterol efflux and activating lipoproteinlipase can be used to prevent, treat or inhibit a dyslipidemic orvascular disorder in a subject without causing a substantial cytotoxiceffect. A method of treating or inhibiting a dyslipidemic or vasculardisorder in a subject includes administering to the subject atherapeutically effective amount of the pharmaceutical compositionincluding one or more of the peptides disclosed herein. Atherapeutically effective amount of the pharmaceutical composition isone that results in triglyceride levels of less than 150 mg/dl whilecausing lipid efflux. In a particular example, the pharmaceuticalcomposition is a peptide that is capable of causing by lipid efflux andactivating lipoprotein lipase (including a peptide with an amino acidsequence provided by any one of SEQ ID NOs: 70-75), thereby resulting intriglyceride levels of less than 150 mg/dl. For example, apharmaceutical composition including a peptide with the amino acidsequence of SEQ ID NO: 70 is provided at a therapeutically effectiveamount to a subject so that triglyceride levels in the subject are 150mg/dl or less, such as between 150 mg/dl and 10 mg/dl, 140 mg/dl and 25mg/dl, 120 mg/dl and 50 mg/dl, including 140 mg/dl, 130 mg/dl, 120mg/dl, 110 mg/dl, 100 mg/dl, 90 mg/dl, 80 mg/dl, 70 mg/dl, 60 mg/dl, 50mg/dl, 40 mg/dl, 30 mg/dl, 20 mg/dl, or 10 mg/dl, while plasmacholesterol levels are reduced by at least 20%, such as at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% percent, including22%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98%.

In another particular example, the pharmaceutical composition includesat least two peptides, one that is capable of causing lipid efflux andanother that is capable of activating lipoprotein lipase. In a certainexample, a pharmaceutical composition including a peptide with an aminoacid sequence set forth in any one of SEQ ID NOs: 3-45 and an apoC-IIpeptide with an amino acid sequence set forth in any one of SEQ ID NOs:63-69. These peptides need not be covalently attached one to another. Ina specific example, the pharmaceutical composition includes a peptidewith an amino acid sequence set forth in SEQ ID NO: 3 and an apoC-IIpeptide with an amino acid sequence set forth SEQ ID NO: 63. Thispharmaceutical composition is provided at a therapeutically effectiveamount to a subject so that triglyceride levels are 150 mg/dl or less,such as between 150 mg/dl and 10 mg/dl, 140 mg/dl and 25 mg/dl, 120mg/dl and 50 mg/dl, including 140 mg/dl, 130 mg/dl, 120 mg/dl, 110mg/dl, 100 mg/dl, 90 mg/dl, 80 mg/dl, 70 mg/dl, 60 mg/dl, 50 mg/dl, 40mg/dl, 30 mg/dl, 20 mg/dl, or 10 mg/dl, while plasma cholesterol levelsare reduced by at least 20%, such as at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% percent, including 22%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%.

It will be apparent that the precise details of the constructs,compositions, and methods described herein may be varied or modifiedwithout departing from the spirit of the described invention. We claimall such modifications and variations that fall within the scope andspirit of the claims below.

1. An isolated peptide or peptide analog, comprising: a first domain capable of promoting lipid efflux from cells by an ABCA1-dependent pathway; and a second domain capable of activating lipoprotein lipase, wherein the first domain comprises amino acids 1-18 of any one of SEQ ID NOs: 70-75 and the second domain comprises an apoC-II protein, or a variant or fragment thereof which activates lipoprotein lipase thereby promoting lipid efflux without inducing hypertriglyceridemia.
 2. (canceled)
 3. The isolated peptide or peptide analog of claim 1, wherein the second domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 63-68.
 4. The isolated peptide or peptide analog of claim 1, further comprising a linker connecting the first domain to the second domain.
 5. The isolated peptide or peptide analog of claim 4, wherein the linker comprises glycine, alanine or proline.
 6. The isolated peptide or peptide analog of claim 5, wherein the linker comprises proline.
 7. The isolated peptide or peptide analog of claim 1, further comprising at least one additional peptide domain.
 8. The isolated peptide or peptide analog of claim 8, wherein the additional peptide domain comprises a heparin binding site, an integrin binding site, a P-selectin site, a TAT HIV sequence, a panning sequence, a penatratin sequence, a SAA C-terminus sequence, a SAA N-terminus sequence, a LDL receptor sequence, a modified 18A sequence, an apoA-I Milano sequence, a 6×-His sequence, a lactoferrin sequence, or combinations of two or more thereof.
 9. The isolated peptide or peptide analog of claim 1, wherein the peptide or peptide analog comprises an amino acid sequence as set forth in any one of SEQ. ID NOs: 70-75.
 10. An isolated nucleic acid molecule encoding any one of the peptides or peptide analogs according to claim
 9. 11. A pharmaceutical composition, comprising the isolated peptide or peptide analog of claim 1, or an isolated nucleic acid molecule encoding the isolated peptide or peptide analog of claim 1, and a pharmaceutically acceptable carrier.
 12. A method of treating or inhibiting a dyslipidemic or vascular disorder in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim
 11. 13. The method of claim 12, wherein the dyslipidemic or vascular disorder comprises hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoA-I deficiency, coronary artery disease, atherosclerosis, thrombotic stroke, peripheral vascular disease, restenosis, acute coronary syndrome, post-perfusion myocardial injury, vasculitis, inflammation, or combinations of two or more thereof.
 14. (canceled)
 15. The method of claim 13, further comprising administering an additional lipid lowering composition.
 16. The method of claim 12, wherein the pharmaceutical composition is delivered on an implant. 17-18. (canceled)
 19. An implant coated with the peptide or peptide analog of claim
 1. 20. The implant of claim 19, wherein the peptide or peptide analog further comprises at least one additional peptide domain.
 21. The implant of claim 20, wherein the additional peptide domain comprises a heparin binding site, an integrin binding site, a P-selectin site, a TAT HIV sequence, a panning sequence, a penatratin sequence, a SAA C-terminus sequence, a SAA N-terminus sequence, a LDL receptor sequence, a modified 18A sequence, an apoA-I Milano sequence, a 6×-His sequence, a lactoferrin sequence, or combinations of two or more thereof.
 22. The implant of claim 19 which, when implanted in a heart or peripheral vasculature, treats or inhibits a dyslipidemic or vascular disorder.
 23. The peptide of claim 1 for use in the manufacture of an implant for treating or inhibiting a dyslipidemic or vascular disorder.
 24. A method of treating or inhibiting a dyslipidemic or vascular disorder in a subject, comprising co-administering to the subject a therapeutically effective amount of an isolated peptide or peptide analog comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3-45 (which promotes lipid efflux) and an isolated peptide or peptide analog comprising an amino acid sequence set forth in any one of SEQ ID NOs: 63-69 (which activates lipoprotein lipase), thereby treating or inhibiting the dyslipidemic or vascular disorder in the subject.
 25. The method of claim 24, wherein the lipid efflux promoting isolated peptide or peptide analog and the lipoprotein lipase activating peptide or peptide analog are administered within the same delivery vehicle. 26-27. (canceled) 